Transgenic expression cassettes for expressing nucleic acid sequences in sink tissues of plants that store carbohydrate

ABSTRACT

The invention relates to methods for the directed, transgenic expression of nucleic acid sequences in the carbohydrate-storing sink tissues of plants using transgenic expression cassettes which comprise the  Vicia faba  plastidic 1,4-α-D-glucan:phosphate α-D-glucosyltransferase promoter. Furthermore, the invention relates to said transgenic expression cassettes and to transgenic expression vectors and transgenic organisms comprising them, and to the use of the same for the production of foodstuffs, feedstuffs, seed, pharmaceuticals or fine chemicals.

RELATED APPLICATIONS

This application is a national stage application (under 35 U.S.C. 371)of PCT/EP2003/009855 filed Sep. 5, 2003, which claims benefit of Germanapplication 102 42 204.4 filed Sep. 10, 2002.

The invention relates to methods for the directed, transgenic expressionof nucleic acid sequences in the carbohydrate-storing sink tissues ofplants using transgenic expression cassettes which comprise the Viciafaba plastidic 1,4-α-D-glucan:phosphate α-D-glucosyltransferasepromoter. Furthermore, the invention relates to said transgenicexpression cassettes and to transgenic expression vectors and transgenicorganisms comprising them, and to the use of the same for the productionof foodstuffs, feedstuffs, seed, pharmaceuticals or fine chemicals.

The aim of plant biotechnology work is the generation of plants withimproved properties, for example for increasing agriculturalproductivity. Transcriptional regulatory sequences or promoters whichregulate the expression of genes in plants are essential elements ofplant biotechnology. Various promoters which have been used successfullyfor the expression of heterologous genes in plants are available; theycomprise not only plant promoters (such as, for example, cauliflowerheat shock protein hsp80 promoters; U.S. Pat. No. 5,612,472), but alsopromoters from other non-plant sources such as, for example, promotersof plant viruses (for example the cauliflower mosaic virus 35S promoter)or of plant-infecting bacteria (for example the promoter of theagrobacterium octopine synthase; Leisner and Gelvin (1988) Proc NatlAcad Sci USA 85(8):2553-2557).

Frequently, what are known as constitutive promoters, which regulate, inthe plant, the expression of a gene product largely at any time and inany tissue are employed for the expression of heterologous nucleic acidsequences in transgenic plants. A directed expression of genes inspecific plant parts or at specific points in time of the development isnot possible using these promoters. Thus, the protein to be expressedtransgenically is expressed at locations and at times where it is notrequired, which, for example, unnecessarily consumes energy, causesmetabolic modifications and can thus have an adverse effect on plantgrowth. For reasons of product licensing and product acceptance too, itis desirable to express a transgenic protein only where it is requiredowing to its intended effect.

Tissue- and development-specific promoters are of great interest forthis purpose. Various such promoters are known. Thus, the promoter ofthe Vicia faba “sucrose-binding-protein-like gene” (SBP) mediates strongand specific expression in seeds of oilseed rape and other plants (WO00/26388).

Fruits, seeds, beets/swollen tap roots or tubers, being importantstorage organs of plant organisms, are of great agronomical relevance.They serve for the storage of proteins, oils and carbohydrates (inparticular starch). As a rule, such tissues are photosyntheticallyinactive and are also referred to as sink tissues or sink organs. Theyrely on the import of photoassimilates from the photosyntheticallyactive plant parts (source organs or source tissues). Both traditionalbreeding and biotechnological methods have been used for improvingspecific aspects of fruit and tuber quality. High-quality, mature fruitsare the result of a number of coordinated biochemical and metabolicmodifications which can occur not only during maturation, but alsoduring fruit development. These modifications determine the finalquality and the quantity of the fruits. Examples of modified properties,for example in the case of tomato fruits, are increased sucrose import,conversion into starch, accumulation of various organic acids,modifications of pigments and modifications in fungicidal andinsecticidal compounds. Such results can be achieved by means of theoverexpression of genes/proteins or by inhibition by means ofdouble-stranded RNA, antisense RNA or cosuppression. Since sink tissuesact as storage site of the most important plant raw materials, promoterswhich make possible a selective expression in these tissues are ofparticular interest in plant biotechnology since they permit a directedmodification of these tissues and of their constituents.

The skilled worker is familiar with a variety of promoters which can beused for the expression of nucleic acid sequences in fruits, seeds ortubers. The promoter of the tomato genomic clone 2A11 must be mentioned(Pear et al. (1989) Plant Mol Biol 13:639-651; Wo 91/19806). However,the 2A11 promoter governs expression during the very early stages and isrelatively weak. The tomato ethylene-inducible E4 and E8 promoters (U.S.Pat. No. 5,859,330; Deickmann et al. (1988) EMBO J. 7:3315-3320) and thepolygalacturonase promoter (U.S. Pat. No. 6,127,179) have likewise beendescribed as being fruit-specific. The abovementioned promoters,however, show expression only during the late phases of fruitdevelopment, and their use is therefore only limited. The promoters TFM7and TFM9 (U.S. Pat. No. 5,608,150) are active during fruit developmentin green and yellow stages. The fruit-specific regulation of the kiwifruit actinidin promoter has been detected for expression in petunia(Lin et al. (1993) Plant Mol Biol 23:489-499). Thi-1, MADS2 and apromoter fusion between Thi-1 and the melon actin promoter regulate theexpression of heterologous genes specifically in apples (WO 00/66610).

Further promoters are, for example, promoters with specificity fortubers, storage roots or other roots such as, for example, thetuber-specific patatin class I promoter (Bevan et al. (1986) Nucl AcidsRes 14:4625-4638), the potato cathepsin D inhibitor promoter, the starchsynthase (GBSS1) promoter or the sporamin promoter. Other genes withspecific high activity in tubers are, for example, the promoter of theADP-glucose pyrophosphorylase gene (Müller et al. (1990) Mol Gen Genet224:136-146), of sucrose synthase (Salanoubat and Belliard (1987) Gene60:47-56; Salanoubat and Belliard (1989) Gene 84:181-185), the promotersof the 22 kD protein complex and of the proteinase inhibitor (Hannapel(1990) Plant Physiol 94:919-925) and the other class I patatins (B33)(EP-A1 0 375 092; Rocha-Sosa et al. (1989) EMBO J. 8:23-29). Adisadvantage of the patatin 1 promoter is that it is induced by highsucrose concentrations, also in tissues other than the tuber (Jefferson,R. et al. (1990) Plant Mol Biol 14:995-1006).

Glucan phosphorylases (systematic name: 1,4-α-D-glucan:phosphateα-D-glucosyltransferase; frequently also starch phosphorylase; EC2.4.1.1) are found in all organisms which are capable of storing starchor glycogen. The enzyme cleaves terminal α-1,4-linked glucose residuesfrom glucan-like molecules with formation of glucose-1-phosphate. Twoenzymes exist in plant cells which differ with regard to theirlocalization and specificity for glucans. The Pho2 isoform is located inthe cytosol and has high affinity with branched polyglucans such assoluble starch or glycogen. The isoform Pho1 is localized in the stromaof the plastids and prefers unbranched glucan-like amylose andmaltodextrins as substrate. Two homologous genes of this isoform with81% identity have been found in potatoes, the first being expressedpredominantly in tubers and the second in leaves (Sonnewald et al.(1995) Plant Mol Biol 27:567-576). Also, two isoforms were isolated fromthe field bean, Vicia faba (Pho1 and Pho2; Buchner P et al. (1996)Planta 199:64-73). The plastidic isoform Pho1 is probably involved inthe storage of starch biosynthetic pathway and is expressed to a highdegree in essentially the late stages of field bean seed development.Despite the large number of studies into structural and kineticproperties of the plant glucan phosphorylases and their distribution inthe various tissues, their precise role in carbohydrate metabolism isunclear. The promoter of a potato gene which encodes a protein with 75%homology to the Vicia faba glucan phosphorylase is described (St-PierreB & Brisson N (1995) Plant Science 110:193-203; St-Pierre et al. (1996)Plant Mol Biol 30:1087-1098). Said promoter shows not only activity inthe tubers, but also an activity in the roots which is up to 1.5 timeshigher than in the tubers. Likewise, high activity was found in thepetioles and in the shoot. In total, the activity in the tubers was lessthan in petioles, in the shoot, in the stolons and in the roots.

The promoters described in the prior art have one or more of thefollowing disadvantages:

-   1) The promoters do not show the desired expression level and/or are    active in a few plant species only.-   2) The promoters are only active very early or very late during    fruit or tuber development.-   3) The expression pattern does not agree with what has been    expected, i.e. for example undesired expression activities in other    tissues are found.-   4) The expression of many of the abovementioned promoters is    ethylene-dependent.

Moreover, the number of existing promoters is greatly limited. This maybecome a limiting factor, in particular in approaches which require theexpression of more than one heterologous nucleic acid sequence. Theexpression, under the same promoter, of different heterologoussequences, in one plant organism can result in “switching off”(“epigenic silencing”) of the transgenic expression cassettes inquestion (Mette et al. (1999) EMBO J. 18:241-248).

It was therefore an object to provide novel promoters and transgenicexpression cassettes derived therefrom which have high specificity forsink tissues or sink organs and high activity over as long as possible adevelopment period. This object is achieved by the present invention.

A first subject of the invention relates to methods for the directed,transgenic expression of nucleic acid sequences in carbohydrate-storingsink tissues of plants, which comprises the following steps:

-   I. Introducing, into plant cells, a transgenic expression cassette,    where the transgenic expression cassette comprises at least the    following elements:    -   a) at least one promoter sequence of the gene encoding the Vicia        faba plastidic 1,4-α-D-glucan:phosphate α-D-glucosyltransferase,        and    -   b) at least one further nucleic acid sequence, and    -   c) if appropriate, further genetic control elements,    -   where at least one of said promoter sequences and one further        nucleic acid sequence are functionally linked with one another        and the further nucleic acid sequence is heterologous with        regard to the promoter sequence, and-   II. selection of transgenic cells which comprise said expression    cassette stably integrated into the genome, and-   III. regeneration of intact plants from said transgenic cells, where    at least one of the further nucleic acid sequence is expressed in    carbohydrate-storing sink tissue, but essentially not in source    tissues.

A further subject relates to transgenic expression cassettes as can beused for example in the method according to the invention. Preferably,the transgenic expression cassettes for the directed, transgenicexpression of nucleic acid sequences in the carbohydrate-storing sinktissues of plants comprise

-   a) at least one promoter sequence of the gene encoding the Vicia    faba plastidic 1,4-α-D-glucan:phosphate α-D-glucosyltransferase, and-   b) at least one further nucleic acid sequence, and-   c) if appropriate, further genetic control elements,    where at least one promoter sequence and one further nucleic acid    sequence are functionally linked with one another and the further    nucleic acid sequence is heterologous with regard to the promoter    sequence.

In a preferred embodiment of the method according to the inventionand/or the expression cassettes according to the invention, “promotersequence of a gene encoding the Vicia faba plastidic1,4-α-D-glucan:phosphate α-D-glucosyltransferase” means promotersequences which comprise at least one sequence selected from the groupof sequences consisting of

-   i) the promoter sequence of SEQ ID NO: 1 and-   ii) functionally equivalent promoter sequences which have at least    40% homology with the sequence of SEQ ID NO: 1 over a sequence    segment of at least 100 base pairs and which have essentially the    same promoter activity as the promoter sequence of SEQ ID NO: 1, and-   iii) functionally equivalent fragments of the promoter sequence    of i) or ii) with a length of at least 100 base pairs and    essentially the same promoter activity as the promoter sequence of    SEQ ID NO: 1.

The expression cassettes according to the invention may comprise furthergenetic control sequences and/or additional functional elements.

Preferably, the transgenic expression cassettes can make possible, owingto the nucleic acid sequence to be expressed transgenically, theexpression of a protein encoded by said nucleic acid sequence and/or theexpression of a sense RNA, antisense RNA or double-stranded RNA encodedby said nucleic acid sequence.

A further subject of the invention relates to transgenic expressionvectors which comprise one of the expression cassettes according to theinvention.

A further subject of the invention relates to transgenic organisms whichcomprise one of the expression cassettes or expression vectors accordingto the invention. The organism can be selected from the group consistingof bacteria, yeasts, fungi, nonhuman animal organisms and plantorganisms or cells, cell cultures, parts, tissues, organs or propagationmaterial derived therefrom; preferably, the organism is selected fromthe group of the agricultural useful plants. In said plants, theexpression of the nucleic acid sequence to be expressed transgenicallyis preferably higher in at least one sink tissue, preferably in acarbohydratestoring sink tissue (for example the potato tuber or thetomato fruit) than in another tissue.

A further subject of the invention therefore relates to an isolatednucleic acid sequence comprising the promoter of the Vicia fabaplastidic 1,4-α-D-glucan:phosphate α-D-glucosyltransferase of SEQ ID NO:1 or a functionally equivalent fragment of same with essentially thesame promoter activity.

In a preferred embodiment, the nucleic acid sequence according to theinvention, or the transgenic expression cassette according to theinvention in the form of a functionally equivalent promoter sequence,additionally comprises the sequence encoding the 5′-untranslated regionof the Vicia faba Pho1 gene in addition to the sequence of SEQ ID NO: 1.Especially preferred is the sequence described by SEQ ID NO: 2.

In a further preferred embodiment, the nucleic acid sequence accordingto the invention, or the transgenic expression cassette according to theinvention in the form of a functionally equivalent promoter sequence,additionally comprises, in addition to the sequence of SEQ ID NO: 1, thesequence encoding the 5′-untranslated region of the Vicia faba Pho1 geneand a sequence encoding a transit peptide, preferably the transitpeptide of the Vicia faba Pho1 protein of SEQ ID NO: 8. This sequence ispreferably in 3′-orientation relative to one of the promoters accordingto the invention. Especially preferred as promoter sequence in thiscontext is the sequence described by SEQ ID NO: 3.

A further subject relates to the use of the isolated nucleic acidsequences, transgenic expression vectors or transgenic organismsaccording to the invention for the transgenic expression of nucleicacids and/or proteins. Especially preferred is the use of saidtransgenic organisms or of cells, cell cultures, parts, tissues, organsor propagation material derived therefrom for the production offoodstuffs, feedstuffs, seed, pharmaceuticals or fine chemicals, thefine chemicals preferably being enzymes, vitamins, amino acids, sugars,saturated or unsaturated fatty acids, natural or synthetic flavorings,aroma substances or colorings. Furthermore comprised by the inventionare methods for the production of said foodstuffs, feedstuffs, seed,pharmaceuticals or fine chemicals using the transgenic organismsaccording to the invention or cells, cell cultures, parts, tissues,organs or propagation material derived therefrom.

Surprisingly, the promoter of the plastidic 1,4-α-D-glucan:phosphateα-D-glucosyltransferase (hereinbelow “Pho1 promoter”) from the fieldbean Vicia faba shows high-level, selective expression incarbohydrate-storing sink tissues, such as the potato tubers,beets/swollen tap roots and in fruits, for example tomato. Thus, forexample, high-level expression can be found in the potato tuber andtomato fruit, while no discernible expression activity was detected inthe seeds of oilseed rape or Arabidopsis. The expression activitycorrelates to a surprising degree with the location/extent of starchbiosynthesis or the starch content.

The promoter according to the invention has no homology whatsoever withthe known promoter of a putative starch phosphorylase from potato(St-Pierre B & Brisson N (1995) Plant Science 110:193-203; St-Pierre etal. (1996) Plant Mol Biol 30:1087-1098). In addition to an activity inthe tubers, the potato starch phosphorylase promoter has an activity inthe roots which is up to 1.5 times higher than in the tubers. Likewise,high activities were found in the petioles and in the shoot. In total,the activity in the tubers was less than in petioles, in the shoot, inthe stolons and in the roots. In contrast, the Vicia faba Pho1 promoteraccording to the invention has the advantage that, besides thesurprisingly high activity in the potato tubers and in the tomatofruits, only very low activities, or none, were found in other tissues.FIG. 2 shows the tissue-specific expression of the Pho1 promoter duringfruit development in tomatoes. Only little activity was detected in theleaves and the roots. The promoter is at its most active in young greenfruits; this activity decreases as the fruit ripens.

The USP promoter (Baumlein et al. (1991) Mol Gen Genet 225:459-467) wasstudied in comparison. Surprisingly, β-glucuronidase (GUS) expressionexperiments showed high-level expression in the potato tubers and in thetomato fruits for the Pho1 promoter only, while no expression was foundfor the USP promoter. Accordingly, there is no inherent linkage betweenexpression in seeds and carbohydrate-storing sink tissue.

Expression, brought about by the Pho1 promoter, is high, in particularin immature, green fruits, and decreases in the maturing, red fruits.Here, the expression level correlates with the starch content (cf. FIG.2 vs. FIG. 4). A similar correlation can also be found in potatoes, butin this case the starch is accumulated only when the tuber has reachedthe adult stage, and it is here that the promoter shows its maximumactivity. No significant expression was detected in other tissues.

Owing to its high expression activity and its high specificity, the Pho1promoter according to the invention is particularly valuable for plantbiotechnology. It can be expected that the Pho1 promoter will also beactive in carbohydrate- and/or storage-starch-containing tissues ofother plants. In particular the fact that its activity has an earlyonset during organ development can be exploited advantageously forincreasing the quality of the developing fruit. Very especiallyadvantageous in this context is the use in approaches which serve forthe modification of carbohydrate and/or starch biosynthesis or of thecarbohydrate and/or starch metabolism. Examples of nucleic acidsequences to be expressed by preference in this context are givenhereinbelow.

“Expression” means the transcription of the nucleic acid sequence to beexpressed transgenically, but can—in the case of an open reading framein sense orientation—also include the translation of the transcribedRNA, of the nucleic acid sequence to be expressed transgenically, into acorresponding polypeptide.

“Transgenic” means—for example regarding a transgenic expressioncassette, a transgenic expression vector, a transgenic or ganism ormethod for the transgenic expression of nucleic acids all thoseconstructs which are the result of transgenic methods, or all methodsusing them, in which either

-   a) the Pho1 promoter of SEQ ID NO: 1, 2 or 3 a functionally    equivalent fragment of the above, or-   b) the nucleic acid sequence to be expressed transgenically, in    functional linkage with a promoter of a), or-   c) (a) and (b)    are not located in their natural genetic environment or have been    modified by transgenic methods, where the modification can be for    example a substitution, addition, deletion, inversion or insertion    of one or more nucleotide residues. Preferably, the promoter    sequence according to the invention which is present in the    expression cassettes (for example the sequence of SEQ ID NO: 1, 2    or 3) is heterologous with regard to the further nucleic acid    sequence which is linked functionally with it and which is to be    expressed transgenically. In this context, “heterologous” means that    the further nucleic acid sequence does not encode the gene which is    naturally under the control of said promoter.

“Natural genetic environment” means the natural chromosomal locus in theorganism of origin or the presence in a genomic library. In the case ofa genomic library, the natural genetic environment of the nucleic acidsequence is preferably retained at least in part. The environment flanksthe nucleic acid sequence at least at one side and has a sequence lengthof at least 50 bp, preferably at least 500 bp, especially preferably atleast 1000 bp, very especially preferably at least 5000 bp. A naturallyoccurring expression construct—for example the naturally occurringcombination of the promoter of SEQ ID NO: 1 and the coding sequence ofthe 1,4-α-D-glucan:phosphate α-D-glucosyltransferase gene from the fieldbean Vicia faba becomes a transgenic expression construct when thiscombination is modified by normatural, synthetic (“artificial”) methodssuch as, for example, an in-vitro mutagenesis. Such methods have beendescribed (U.S. Pat. No. 5,565,350; WO 00/15815; see also hereinabove).

“Transgenic” with regard to an expression (“transgenic expression”)preferably means all those expressions which have been carried out usinga transgenic expression cassette, transgenic expression vector ortransgenic organism, as defined hereinabove.

“Directed” with regard to the expression in carbohydrate-storing sinktissues means that the expression under the control of one of thepromoters according to the invention in the carbohydratestoring sinktissues amounts to preferably at least ten times, very especiallypreferably to at least fifty times, most preferably to at least hundredtimes the expression level in another tissue, preferably in a sourcetissue such as, for example, the leaves.

The transgenic expression cassettes according to the invention, thetransgenic expression vectors and transgenic organisms derived from themcan comprise functional equivalents of the Pho1 promoter sequencedescribed in SEQ ID NO: 1. Said functional equivalents have at least40%, preferably at least 60%, especially preferably at least 80%, veryespecially preferably at least 90% homology with the sequence of SEQ IDNO: 1 over a sequence segment of at least 100 base pairs, preferably atleast 200 base pairs, very especially preferably at least 500 basepairs, most preferably over the entire sequence length, and haveessentially the same promoter activity as the promoter sequence of SEQID NO: 1.

The transgenic expression cassettes according to the invention, thetransgenic expression vectors and transgenic organisms derived from themcan comprise functionally equivalent fragments of the promoter of SEQ IDNO: 1 or of a functional equivalent of the same. To prepare suchfunctionally equivalent fragments, it is possible, for example, todelete nonessential sequences of a promoter according to the inventionwithout adversely affecting the abovementioned essential properties to asignificant extent. Such deletion variants constitute functionallyequivalent fragments of the Pho1 promoter described by SEQ ID NO: 1 orof a functional equivalent of the same. The delimitation of the promotersequence to certain, essential regulatory regions can be carried out forexample with the aid of search routines for the search of promoterelements. Frequently, certain promoter elements are amassed in theregions which are relevant for the promoter activity. This analysis canbe carried out for example with computer programs such as the programPLACE (“Plant Cis-acting Regulatory DNA Elements”) (Higo K et al. (1999)Nucl Acids Res 27(1): 297-300) or the BIOBASE database “Transfac”(Biologische Datenbanken GmbH, Braunschweig).

Preferably, the functionally equivalent fragments of one of thepromoters according to the invention—for example the Pho1 promoterdescribed by SEQ ID NO: 1 or of a functional equivalent thereof—compriseat least 100 base pairs, very especially preferably at least 200 basepairs, most preferably at least 500 base pairs of the Pho1 promoterdescribed by SEQ ID NO: 1 or of a functional equivalent thereof. In apreferred embodiment, the functionally equivalent fragment comprises the3′ region of the Pho1 promoter described by SEQ ID NO: 1 or of afunctional equivalent thereof, the fragment length which is in each casepreferred being calculated in 5′ direction upstream from thetranscription start or translation start (“ATG” codon).

A promoter activity is referred to as essentially the same when thetranscription of a certain gene to be expressed under the control of,for example, a functional equivalent fragment of the Pho1 promotersequence described by SEQ ID NO: 1 under otherwise unchangedconditions—in at least one sink tissue, preferably in acarbohydrate-storing, -sythesizing or -metabolizing sink tissue (suchas, for example, the potato tuber, beet and tomato fruit), veryespecially preferably in a starch-storing, -sythesizing or -metabolizingsink tissue (such as, for example, the potato tuber), is higher than inanother tissue, for example a source tissue.

In this context, “carbohydrate” preferably means starch or sucrose,especially preferably starch.

“Source tissue” means photosynthetically active tissue. “Sink tissue”means tissues which are net importers of photosynthetically fixed carbondioxide and which, as a rule, are not photosynthetically active.Examples of sink tissues which may be mentioned are: roots, fruits,tubers and seed kernels.

Starch-storing sink tissue (hereinbelow “starch sink tissue”) preferablymeans those tissues which

-   a) are not photosynthetically active themselves and-   b) have at at least one point in time of their development a starch    content which can be detected by means of a starch detection    reaction. A preferred starch detection reaction is staining with    Lugol's solution (Lugol's solution: for example: dissolve 2 g of KI    in 5 ml of water, dissolve 1 g of iodine in this solution and add    300 ml of water). Staining, is carried out until a discernible blue    coloration has appeared (approximately 15 minutes at RT) and can be    stopped by washing with water.

Here, the expression under the control of one of the promoters accordingto the invention in a carbohydrate-storing, -synthesizing or-metabolizing sink tissue or a starch sink tissue is preferably at leasttwice, very especially preferably at least five times, most preferablyat least ten times as high as in another tissue, for example a sourcetissue.

Sequences which are preferably employed when determining the expressionlevel are those which code readily quantifiable proteins. Veryespecially preferred in this context are reporter proteins (Schenborn E,Groskreutz D. (1999) Mol Biotechnol 13(1): 29-44) such as “greenfluorescence protein” (GFP) (Chui W L et al. (1996) Curr Biol 6:325-330;Leffel S M et al. (1997) Biotechniques 23(5):912-8), chloramphenicoltransferase, luciferase (Millar et al. (1992) Plant Mol Biol Rep10:324-414), β-glucuronidase or β-galactosidase. β-Glucuronidase(Jefferson et al. (1987) EMBO J. 6:3901-3907) is very especiallypreferred.

“Otherwise unchanged conditions” means that the expression which isinitiated by one of the transgenic expression cassettes to be comparedis not modified by combination with additional genetic controlsequences, for example enhancer sequences. Unchanged conditionsfurthermore means that all framework conditions such as, for example,plant species, developmental stage of the plants, growing conditions,assay conditions (such as buffers, temperature, substrates and the like)are kept identical between the expressions to be compared.

The expression level of a functionally equivalent promoter can deviateboth downward and upward compared with the promoter of SEQ ID NO: 1.Preferred in this context are those sequences whose expression level,measured on the basis of the transcribed mRNA or the subsequentlytranslated protein, under conditions which are otherwise unchangeddiffers quantitatively by not more than 50%, preferably 25%,particularly preferably 10%, from a comparison value obtained with thosepromoters described by SEQ ID NO: 1. Especially preferred sequences arethose whose expression level, measured on the basis of the transcribedmRNA or the subsequently translated protein, under conditions which areotherwise unchanged exceeds quantitatively a comparison value obtainedwith the promoter described by SEQ ID NO: 1 by more than 50%, preferably100%, especially preferably 500%, very especially preferably 1000%.Preferred as comparison value is the expression level of the mRNAs of a1,4-α-D-glucan:phosphate α-D-glucosyltransferase expressed naturally bythe promoter, or the protein resulting therefrom. Furthermore preferredas comparison value is the expression level obtained with any definednucleic acid sequence, preferably those nucleic acid sequences whichencode readily quantifiable proteins. Very especially preferred in thiscontext are reporter proteins (Schenborn E & Groskreutz D (1999) MolBiotechnol 13(1):29-44) such as the “green fluorescence protein” (GFP)(Chui W L et al. (1996) Gurr Biol 6:325-330; Leffel S M et al. (1997)Biotechniques. 23(5):912-8), the chloramphenicol transferase, aluciferase (Millar et al. (1992) Plant Mol Biol Rep 10:324-414) or theβ-glucuronidase; β-glucuronidase is very especially preferred (Jeffersonet al. (1987) EMBO J. 6:3901-3907).

Functional equivalents also comprise natural or artificial mutations ofthe promoter sequence described in SEQ ID NO: 1. Mutations comprisesubstitutions, additions, deletions, inversions or insertions of one ormore nucleotide residues. Thus, for example, the present invention alsocomprises those nucleotide sequences which are obtained by modificationof the Pho1 promoter of SEQ ID NO: 1. The aim of such a modification maybe the further delimitation of the sequence present therein or else, forexample, the introduction or removal of restriction endonucleasecleavage sites, the removal of superfluous DNA or the addition offurther sequences, for example further regulatory sequences.

Where insertions, deletions or substitutions such as, for example,transitions and transversions, are suitable it is possible to usetechniques known per se, such as in-vitro mutagenesis, primer repair,restriction or ligation. Transition means a base pair exchange of apurine/pyrimidine pair into another purine/pyrimidine pair (for exampleA-T for G-C). Transversion means a base pair exchange of apurine/pyrimidine pair for a pyrimidine/purine pair (e.g. A-T for T-A).Deletion means removal of one or more base pairs. Insertion means theintroduction of one or more base pairs.

Complementary ends of the fragments can be made available for ligationby means of manipulations such as restriction, chewing back or fillingin overhangs for what are known as blunt ends. Analogous results canalso be obtained using the polymerase chain reaction (PCR) usingspecific oligonucleotide primers.

Homology between two nucleic acids is understood as meaning the identityof the nucleic acid sequence over the complete sequence length in eachcase, which is calculated by comparison with the aid of the GAP programalgorithm (Wisconsin Package Version 10.0, University of Wisconsin,Genetics Computer Group (GCG), Madison, USA), setting the followingparameters:

Gap Weight: 12 Length Weight: 4 Average Match: 2.912 Average Mismatch:−2.003

For example, a sequence which has at least 50% homology with thesequence SEQ ID NO: 1 on nucleic acid basis is understood as meaning asequence which, upon comparison with the sequence SEQ ID NO: 1 by theabove program algorithm with the above set of parameters has at least50% homology.

Functional equivalents also means DNA sequences which hybridize understandard conditions with the nucleic acid sequence encoding the Pho1promoter as shown in SEQ ID NO: 1 or with the nucleic acid sequencescomplementary thereto, and which have substantially the same promoterproperties. The term standard hybridization conditions is to beunderstood broadly and means both stringent and less stringenthybridization conditions. Such hybridization conditions are describedinter alia in Sambrook J, Fritsch E F, Maniatis T et al., in MolecularCloning—A Laboratory Manual, 2^(nd) edition, Cold Spring HarborLaboratory Press, 1989, pages 9.31-9.57 or in Current Protocols inMolecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.

For example, the conditions during the washing step can be selected fromthe range of conditions limited by those of low stringency (withapproximately 2×SSC at 50° C.) and of high stringency (withapproximately 0.2×SSC at 0.50° C., preferably at 65° C.) (20×SSC: 0.3 Msodium citrate, 3 M NaCl, pH 7.0). In addition, the temperature duringthe washing step can be raised from low-stringency conditions at roomtemperature, approximately 22° C., to more stringent conditions atapproximately 65° C. Both parameters, the salt concentration and thetemperature, can be varied simultaneously, and it is also possible forone of the two parameters to be kept constant and only the other to bevaried. It is also possible to employ denaturing agents such as, forexample, form amide or SDS during the hybridization. Hybridization inthe presence of 50% formamide is preferably carried out at 42° C. Someexemplary conditions for hybridization and washing steps are givenbelow:

-   (1) Hybridization conditions with for example    -   a) 4×SSC at 65° C., or    -   b) 6×SSC, 0.5% SDS, 100 μg/ml denatured fragmented salmon sperm        DNA at 65° C., or    -   c) 4×SSC, 50% formamide, at 42° C., or    -   d) 2× or 4×SSC at 50° C. (low-stringency condition), or    -   e) 2× or 4×SSC, 30 to 40% formamide at 42° C. (low-stringency        condition), or    -   f) 6×SSC at 45° C., or,    -   g) 0.05 M sodium phosphate buffer pH 7.0, 2 mM EDTA, 1% BSA and        7% SDS.-   (2) Washing steps with for example    -   a) 0.1×SSC at 65° C., or    -   b) 0.1×SSC, 0.5% SDS at 68° C., or    -   c) 0.1×SSC, 0.5% SDS, 50% formamide at 42° C., or    -   d) 0.2×SSC, 0.1% SDS at 42° C., or    -   e) 2×SSC at 65° C. (low-stringency condition), or    -   f) 40 mM sodium phosphate buffer pH 7.0, 1% SDS, 2 mM EDTA.

Methods for preparing functional equivalents of the invention preferablycomprise the introduction of mutations into the Pho1 promoter as shownin SEQ ID NO: 1. Mutagenesis may be random, in which case themutagenized sequences are subsequently screened for their properties bya trial and error procedure. Particularly advantageous selectioncriteria comprise for example the level of the resulting expression ofthe introduced nucleic acid sequence in a starch-sink tissue.

Methods for mutagenesis of nucleic acid sequences are known to theskilled worker and include by way of example the use of oligonucleotideswith one or more mutations compared with the region to be mutated (e.g.in a site-specific mutagenesis). Primers with approximately 15 toapproximately 75 nucleotides or more are typically employed, withpreferably about 10 to about 25 or more nucleotide residues beinglocated on both sides of the sequence to be modified. Details andprocedure for said mutagenesis methods are familiar to the skilledworker (Kunkel et al. (1987) Methods Enzymol 154:367-382; Tomic et al.(1990) Nucl Acids Res 12:1656; Upender et al. (1995) Biotechniques18(1):29-30; U.S. Pat. No. 4,237,224). A mutagenesis can also beachieved by treating for example transgenic expression vectorscomprising one of the nucleic acid sequences of the invention withmutagenizing agents such as hydroxylamine.

A functional linkage means, for example, the sequential arrangement ofone of the promoters according to the invention, of the nucleic acidsequence to be expressed transgenically and, if appropriate, of furtherregulatory elements such as, for example, a terminator in such a waythat each of the regulatory elements is able to fulfill its function,depending on the arrangement of the nucleic acid sequences in relationto sense or antisense RNA, in the transgenic expression of the nucleicacid sequence. This does not necessarily require a direct linkage in thechemical sense. Genetic control sequences such as, for example, enhancersequences, can also exert their function on the target sequence frompositions which are further removed, or indeed from other DNA molecules.Preferred arrangements are those in which the nucleic acid sequence tobe expressed transgenically is positioned behind the sequence which actsas promoter so that both sequences are mixed covalently with oneanother. Preferably, the distance between the promoter sequence and thenucleic acid sequence to be expressed transgenically is less than 200base pairs, especially preferably less than 100 base pairs, veryespecially preferably less than 50 base pairs.

A transgenic expression cassette or a functional linkage can be producedby means of conventional recombination and cloning techniques as aredescribed, for example, in Maniatis T et al. (1989) Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y. and in Silhavy T J et al. (1984) Experiments with Gene Fusions,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. and in Ausubel FM et al. (1987) Current Protocols in Molecular Biology, GreenePublishing Assoc. and Wiley Interscience. A method which is suitable forthis purpose is, for example, the GATEWAY™ cloning technology(Invitrogen Inc.), which is based on recombination.

A transgenic expression cassette according to the invention is preparedfor example by fusing one of the promoters according to the invention asshown in SEQ ID NO: 1 (or a functional equivalent or functionallyequivalent part thereof) with a nucleotide sequence to be expressedtransgenically, if appropriate with a sequence encoding a transitpeptide, preferably a chloroplast-specific transit peptide, which ispreferably arranged between the promoter and the nucleotide sequence inquestion, and optionally with a terminator or polyadenylation signal.The combination of the Pho1 promoter with the sequence encoding itsnatural transit peptide (corresponding to SEQ ID NO: 3) is especiallypreferred in this context.

However, a transgenic expression cassette also means those constructionsin which one of the promoters of the invention is, without having beenfunctionally linked beforehand to a nucleic acid sequence to beexpressed transgenically, introduced into a host genome, for example bytargeted homologous recombination or random insertion, where itundertakes regulatory control over nucleic acid sequences thenfunctionally linked thereto, and governs the transgenic expressionthereof. Insertion of the promoter—for example by a homologousrecombination—in front of a nucleic acid encoding for a particularpolypeptide results in a transgenic expression cassette of the inventionwhich governs the expression of the particular polypeptide in the plant.Furthermore, the insertion of the promoter may also take place in such away that antisense RNA to the nucleic acid encoding a certainpolypeptide is expressed. Thus, the expression of the particularpolypeptide in plants is downregulated or silenced.

The nucleic acid sequences which are present in the transgenicexpression cassettes according to the invention and which are to beexpressed transgenically can be linked functionally with further geneticcontrol sequences, besides one of the promoters according to theinvention.

The concept of the genetic control sequences is to be understood broadlyand means all those sequences which have an effect on the origin or thefunction of the transgenic expression cassette according to theinvention. Genetic control sequences modify, for example, thetranscription and/or translation in prokaryotic or eukaryotic organisms.Preferably, the transgenic expression cassettes according to theinvention comprise one of the promoters according to the invention5′-upstream from the particular nucleic acid sequence to be expressedtransgenically and a terminator sequence 3′-downstream as additionalgenetic control sequence, and, if appropriate, further customaryregulatory elements, in each case functionally linked with the nucleicacid sequence to be expressed transgenically.

Genetic control sequences also comprise further promoters, promoterelements or minimal promoters which are capable of modifyingexpression-controlling properties. It is thus possible, by means ofgenetic control sequences, that for example tissue-specific expressiontakes place in addition in dependence on certain stress factors.Suitable elements are described, for example, for water stress, abscisicacid (Lam E and Chua N H (1991) J Biol Chem 266(26):17131-17135) andheat stress (Schöffl F et al. (1989) Mol Gen Genet (217(2-3):246-53).

It is furthermore possible that further promoters which make possibleexpression in further plant tissues or in other organisms such as, forexample, E. coli bacteria, are linked functionally with the nucleic acidsequence to be expressed. Suitable plant promoters are, in principle,all of the above-described promoters. For example, it is conceivablethat a certain nucleic acid sequence is transcribed, in a plant tissue,as sense RNA, and translated into the corresponding protein, by onepromoter (for example one of the promoters according to the invention),while the same nucleic acid sequence is, in a different tissue,transcribed into antisense RNA, and the corresponding protein isdownregulated, by a different promoter with a different specificity.This can be carried out by means of a transgenic expression cassetteaccording to the invention by positioning the one promoter before thenucleic acid sequence to be expressed transgenically and the otherpromoter behind it.

Genetic control sequences furthermore also comprise the 5′-untranslatedregion, introns, the noncoding 3′ region or else sequences of genes,preferably the Pho1 gene, which encode signal or transit peptides. Ithas been shown that these regions may have significant functions inregulating gene expression. Thus, it has been shown that 5′-untranslatedsequences are capable of enhancing the transient expression ofheterologous genes. Furthermore, they may promote tissue specificity(Rouster J et al. (1998) Plant J 15:435-440). Conversely, the5′-untranslated region of the opaque-2 gene suppresses expression.Deletion of the region in question results in an increase in geneactivity (Lohmer S et al. (1993) Plant Cell 5:65-73).

In transgenic rice cells, the use of the Act1 intron in combination withthe 35S promoter led to an expression rate which was increased by afactor of ten in comparison with the isolated 35S promoter (McElroy etal. (1991) Mol Gen Genet 231(1):150-160). An optimization with thesequence environment of the translation initiation site of the GUSreporter gene resulted in a four-fold increase in GUS expression intransformed rice cells. A combination of the optimized translationinitiation site and of the Act1 intron resulted in a 40-fold increase inGUS expression by the CaMV35S promoter in transformed rice cells;similar results were obtained with transformed maize cells. In total,the conclusion drawn from the above-described studies was that theexpression vectors based on the Act1 promoter are suitable forcontrolling a sufficiently high-level and constitutive expression offoreign DNA in transformed cells of monocotyledonous plants.

The promoter sequences shown in SEQ ID NO: 2 or 3 comprise the segmentof the Pho1 gene which represents the promoter and the 5′-untranslatedregion up to before the ATG start codon of the Pho1 protein.

The transgenic expression cassette can advantageously comprise one ormore of what are known as enhancer sequences in functional linkage withthe promoter, which make increased transgenic expression of the nucleicacid sequence possible. Additional advantageous sequences can also beinserted at the 3′ end of the nucleic acid sequences to be expressedtransgenically, such as further regulatory elements or terminators. Thenucleic acid sequences to be expressed transgenically can be present asone or more copies in one of the transgenic expression cassettesaccording to the invention.

Control sequences are furthermore understood as meaning those which makepossible homologous recombination or insertion into the genome of a hostorganism, or which permit deletion from the genome. In the case ofhomologous recombination, one of the promoters according to theinvention may be substituted for the natural promoter of a particulargene, for example. One of the promoters according to the inventioncan—as described above—be placed, by means of homologous recombination,before an endogenous target gene to be expressed transgenically, bylinking the promoter with DNA sequences which are for example homologousto endogenous sequences located upstream of the reading frame of thetarget gene. Such sequences are to be understood as genetic controlsequences. Methods such as the cre/lox technology permittissue-specific, and in some circumstances inducible, deletion of thetransgenic expression cassette from the genome of the host organism(Sauer B (1998) Methods (Duluth) 14(4):381-92). Here, certain flankingsequences are added to the target gene (lox sequences), which later makepossible deletion by means of cre recombinase.

To select cells which have successfully undergone homologousrecombination, or else transformation, it is, as a rule, necessaryadditionally to introduce a selectable marker (see hereinbelow).Homologous recombination is a relatively rare event in highereukaryotes, in particular in plants. Random integrations into the hostgenome predominate. One possibility of deleting the randomly integratedsequences, and thus to increase the concentration of cell clones with acorrect homologous recombination, is the use of a sequence-specificrecombination system as described in U.S. Pat. No. 6,110,736.

Polyadenylation signals which are suitable as control sequences areplant polyadenylation signals and—preferably—those which essentiallycorrespond to T-DNA polyadenylation signals from Agrobacteriumtumefaciens. In a particularly preferred embodiment, the transgenicexpression cassette comprises a terminator sequence which is functionalin plants. Terminator sequences which are functional in plants generallymeans those sequences which are capable of bringing about, in plants,the termination of the transcription of a DNA sequence. Examples ofsuitable terminator sequences are the OCS (octopine synthase) terminatorand the NOS (nopalin synthase) terminator. However, plant terminatorsequences are especially preferred. Plant terminator sequences generallyrefers to those sequences which are part of a natural plant gene.Especially preferred in this context is the terminator of the potatocathepsin D inhibitor gene (GenBank Acc. No.: X74985) or the terminatorof the field bean storage protein gene VfLE1B3 (GenBank Acc. No.:Z26489).

These terminators are at least equivalent to the viral or T-DNAterminators described in the prior art.

Transgenic expression, under the control of the Pho1 promoter, of theproteins encoded by the nucleic acid sequences is possible in anydesired cell compartment such as, for example, the endomembrane system,the vacuole and the chloroplasts. By utilizing the secretory pathway,desired glycosylation reactions, especially folding processes, and thelike are possible. The signal peptide sequences required as geneticcontrol sequences for this purpose may either already be provided inindividual transgenic expression cassettes or else be introduced, intothe transgenic expression cassette, jointly with the nucleic acidsequence to be expressed transgenically, by using a suitable cloningstrategy.

Signal or transit peptide sequences which can be used are bothhomologous or heterologous sequences. Additional heterologous sequenceswhich are preferred for functional linkage, but not limited thereto, arefurther targeting sequences for ensuring subcellular localization in theapoplast, in the vacuole, in plastids, in mitochondria, in theendoplasmic reticulum (ER), in the nucleus, in oil bodies or in othercompartments; and translation enhancers such as the tobacco mosaic virus5′ leader sequence (Gallie et al. (1987) Nucl Acids Res 15:8693-8711)and the like. The method for the targeted transportation into theplastids, of proteins which per se are not located in the plastids, isdescribed (Klosgen R B and Weil J H (1991) Mol Gen Genet 225(2):297-304;Van Breusegem F et al. (1998) Plant Mol Biol 38(3):491-496). Preferredsequences are:

-   a) the transit peptide of the Pho1 protein,-   b) the transit peptide of the small subunit (SSU) of    ribulose-bisphosphatecarboxylase (Rubisco ssu) from, for example,    pea, maize or sunflower,-   c) transit peptides derived from genes of plant fatty acid    biosynthesis, such as the transit peptide of the plastidic acyl    carrier protein (ACP), stearyl-ACP desaturase, β-ketoacyl-ACP    synthase or acyl-ACP thioesterase,-   d) the GBSSI (granule-bound starch synthase I) transit peptide,-   e) the transit peptide of the LHCP II genes-   f) the transketolase transit peptide (EP-A1 0 723 017).

The target sequences can be linked with other targeting sequences whichdiffer from the sequences encoding the transit peptide, in order toensure subcellular localization in the apoplast, in the vacuole, inplastids, in mitochondria, in the endoplasmic reticulum (ER), in thenucleus, in oil bodies or in other compartments. Furthermore,translation enhancers such as the tobacco mosaic virus 5′ leadersequence (Gallie et al. (1987) Nucl Acids Res 15:8693-8711) and the likemay be employed.

As already mentioned, the sequence encoding a transit peptide is—in anespecially preferred embodiment—already inserted into the cassette orthe vector for the preparation of a transgenic expression cassette or atransgenic expression vector. Very especially preferably, this involvesthe use of constructs which comprise the Pho1 promoter in linkage with asequence encoding the putative transit peptide of the Pho1 protein (SEQID NO: 8). A promoter construct which is preferably used in this contextis described by SEQ ID NO: 3. To ensure sufficient processing, thisconstruct comprises sequences encoding approximately a further 10 aminoacids behind the transit peptide, thus ensuring efficient processingthereof. In this case, a protein to be expressed transgenically in theplastids would be cloned into the reading frame behind the sequenceencoding the transit peptide, in the manner with which the skilledworker is familiar, which would bring about the transgenic expression ofa chimeric protein with the Pho1 transit peptide.

The skilled worker is familiar with a multiplicity of nucleic acids orproteins whose expression, controlled by the transgenic expressioncassettes according to the invention, is advantageous. Furthermore, theskilled worker is familiar with a multiplicity of genes through whoserepression or deletion, by means of transgenic expression of, forexample, a suitable double-stranded RNA or an antisense RNA,advantageous effects can likewise be obtained. Suitable for the purposesof the present invention are, in particular, those target genes whichplay a role in the sugar or starch metabolism, in sink-source relations,in the balance of organic acids, as flavor components, in the resistanceto biotic stress factors (pathogens, viruses, insects and diseases), inthe resistance to abiotic stress factors (heat, chill, drought, elevatedmoisture, pollutants, UV radiation), in the consistency of the tissuesor in water/pH ratios, in the improvement of food or feedcharacteristics, the improvement of the germination and/or storagecharacteristics, and in the improvement of the growth rate or the yield.

Increasing the starch content is of particular interest especially inthe case of tomatoes or potatoes. A normal tomato consists ofapproximately 80 to 95% of water, while starch—as the actually relevantcomponent for the production of, for example, tomato paste, ketchup—isonly a minor component. Even a small increase in the starch contentwould be of considerable economic importance. During the early stages ofmaturation, the starch content of tomatoes amounts to 20% and is thusmarkedly higher, whereas later during the development it drops owing tothe starch being mobilized and converted into sugars. In potatoes, anincreased starch content has advantageous effects in particular on thedeep-frying properties.

Nucleic acid sequences whose expression under the control of one of thepromoters according to the invention has advantageous effects may bementioned below by way of example, but not by limitation:

-   1. Improved protection of the plant to abiotic stress factors such    as drought, heat or chill, for example by overexpressing antifreeze    polypeptides from Myoxocephalus Scorpius (WO 00/00512),    Myoxocephalus octodecemspinosus, the Arabidopsis thaliana    transcription activator CBF1, glutamate dehydrogenases (WO 97/12983,    WO 98/11240), calcium-dependent protein kinase genes (WO 98/26045),    calcineurins (WO 99/05902), casein kinase from yeast (WO 02/052012),    farnesyltransferases (WO 99/06580; Pei Z M et al. (1998) Science    282:287-290), ferritin (Deak M et al. (1999) Nature Biotechnology    17:192-196), oxalate oxidase (WO 99/04013; Dunwell J M (1998)    Biotechn Genet Eng Rev 15:1-32), DREBlA factor (“dehydration    response element B 1A”; Kasuga M et al. (1999) Nature Biotech    17:276-286), genes of mannitol or trehalose synthesis such as    trehalose-phosphate synthase or trehalose-phosphate phosphatase (WO    97/42326) or by inhibiting genes such as trehalase (WO 97/50561).-   2. Expression of metabolic enzymes for use in the food-and-feed    sector, for example of phytases and cellulases. Especially preferred    are nucleic acids such as the artificial cDNA which encodes a    microbial phytase (GenBank Acc. No.: A19451) or functional    equivalents thereof.-   3. Achieving a resistance, for example to fungi, insects, nematodes    and diseases, by targeted secretion or accumulation of certain    metabolites or proteins. Examples which may be mentioned are    glucosinolates (defense against herbivores), chitinases or    glucanases and other enzymes which destroy the cell wall of    parasites, ribosome-inactivating proteins (RIPS) and other proteins    of the plant resistance and stress reaction as are induced when    plants are wounded or attacked by microbes, or chemically, by, for    example, salicylic acid, jasmonic acid or ethylene, or lysozymes    from nonplant sources such as, for example, T4-lysozyme or lysozyme    from a variety of mammals, insecticidal proteins such as Bacillus    thuringiensis endotoxin, α-amylase inhibitor or protease inhibitors    (cowpea trypsin inhibitor), lectins such as wheatgerm agglutinin,    RNAses or ribozymes. Further examples are nucleic acids which encode    the Trichoderma harzianum chit42 endochitinase (GenBank Acc. No.:    S78423) or the N-hydroxylating, multi-functional cytochrome P-450    (CYP79) protein from Sorghum bicolor (GenBank Acc. No.: U32624), or    functional equivalents of these.

The accumulation of glucosinolates as protection from pests (Rask L etal. (2000) Plant Mol Biol 42:93-113; Menard R et al. (1999)Phytochemistry 52:29-35), the expression of Bacillus thuringiensisendotoxins (Vaeck et al. (1987) Nature 328:33-37) or the protectionagainst attack by fungi, by expression of chitinases, for example frombeans (Broglie et al. (1991) Science 254:1194-1197), is advantageous.Resistance to pests such as, for example, the rice pest Nilaparvatalugens in rice plants can be achieved by expressing the snowdrop(Galanthus nivalis) lectin agglutinin (Rao et al. (1998) Plant J15(4):469-77).

The expression of synthetic cryIA(b) and cryIA(c) genes, which encodelepidoptera-specific Bacillus thuringiensis Δ-endotoxins can bring abouta resistance to insect pests in various plants (Goyal R K et al. (2000)Crop Protection 19(5):307-312).

Further target genes which are suitable for pathogen defense comprise“polygalacturonase-inhibiting protein” (PGIP), thaumatine, invertase andantimicrobial peptides such as lactoferrin (Lee T J et al. (2002) J AmerSoc Horticult Sci 127(2):158-164).

-   4. Expression of genes which bring about an accumulation of fine    chemicals such as of tocopherols, tocotrienols or carotenoids. An    example which may be mentioned is phytoene desaturase. Preferred are    nucleic acids which encode the Narcissus pseudonarcissus photoene    desaturase (GenBank Acc. No.: X78815) or functional equivalents    thereof.-   5. Production of nutraceuticals such as, for example,    polyunsaturated fatty acids (for example arachidonic acid,    eicosapentaenoic acid or docosahexaenoic acid) by expression of    fatty acid elongases and/or desaturases, or production of proteins    with improved nutritional value such as, for example, with a high    content of essential amino acids (for example the high-methionine 2S    albumin gene of the brazil nut). Preferred are nucleic acids which    encode the Bertholletia excelsa high-methionine 2S albumin (GenBank    Acc. No.: AB044391), the Physcomitrella patens Δ6-acyl-lipid    desaturase (GenBank Acc. No.: AJ222980; Girke et al. (1998) Plant J    15:39-48), the Mortierella alpina Δ6-desaturase (Sakuradani et al.    1999 Gene 238:445-453), the Caenorhabditis elegans Δ5-desaturase    (Michaelson et al. 1998, FEBS Letters 439:215-218), the    Caenorhabditis elegans A5-fatty acid desaturase (des-5) (GenBank    Acc. No.: AF078796), the Mortierella alpina Δ5-desaturase    (Michaelson et al. JBC 273:19055-19059), the Caenorhabditis elegans    Δ6-elongase (Beaudoin et al. 2000, PNAS 97:6421-6426), the    Physcomitrella patens Δ6-elongase (Zank et al. 2000, Biochemical    Society Transactions 28:654-657), or functional equivalents of    these.-   6. Production of high-quality proteins and enzymes for industrial    purposes (for example enzymes, such as lipases) or as    pharmaceuticals (such as, for example, antibodies, blood clotting    factors, interferons, lymphokins, colony stimulation factor,    plasminogen activators, hormones or vaccines, as described by Hood E    E, Jilka J M (1999) Curr Opin Biotechnol 10(4):382-6; Ma J K, Vine N    D (1999) Curr Top Microbiol Immunol 236:275-92). For example, it has    been possible to produce recombinant avidin from chicken albumen and    bacterial β-glucuronidase (GUS) on a large scale in transgenic maize    plants (Hood et al. (1999) Adv Exp Med Biol 464:127-47. Review).-   8. Obtaining an increased storability in cells which normally    comprise fewer storage proteins or storage lipids, with the purpose    of increasing the yield of these substances, for example by    expression of acetyl-CoA carboxylase. Preferred nucleic acids are    those which encode the Medicago sativa acetyl-CoA carboxylase    (accase) (GenBank Acc. No.: L25042), or functional equivalents    thereof.

Further examples of advantageous genes are mentioned for example inDunwell J M, Transgenic approaches to crop improvement, J Exp Bot. 2000;51 Spec No; pages 487-96.

Furthermore, it is possible to express functional analogs of theabovementioned nucleic acids or proteins. In this context, functionalanalogs means all those sequences which have essentially the samefunction, i.e. which are capable of exerting the function (for example asubstrate conversion or a signal transduction), as the protein mentionedby way of example. In this context, the functional analog may indeeddiffer with regard to other features. For example, it may have a higheror lower activity or else have further functionalities. Functionalanalogs furthermore means sequences which encode fusion proteinsconsisting of one of the preferred proteins and other proteins, forexample a further preferred protein, or else a signal peptide sequence.

Furthermore, the skilled worker knows that the above-described genesneed not be expressed directly using the nucleic acid sequences encodingthese genes or repress these above-described genes, for example byantisense. It is also possible to use for example artificialtranscription factors of the zinc finger protein type (Beerli R R et al.(2000) Proc Natl Acad Sci USA 97(4):1495-500). These factors attach inthe regulatory regions of the endogenous genes to be expressed or to berepressed and, depending on the design of the factor, bring about anexpression or repression of the endogenous gene. Thus, the desiredeffects can also be achieved by expressing a suitable zinc fingertranscription factor under the control of one of the promoters accordingto the invention.

Likewise, the transgenic expression cassettes according to the inventioncan be employed for the reduction (suppression) of transcription and/ortranslation of target genes by “gene silencing”. Thus, the transgenicexpression cassettes according to the invention can express nucleicacids which bring about PTGS (post transcriptional gene silencing) orTGS (transcriptional silencing) effects and thus a reduction of theexpression of endogenous genes. Said reduction can be achieved forexample by expression of an antisense RNA (EP-A1 0 458 367; EP-A1 0 140308; van der Krol A R et al. (1988) BioTechniques 6(10):658-676; deLange P et al. (1995) Curr Top Microbiol Immunol 197:57-75, inter alia)or of a double-stranded RNA, each of which has homology with theendogenous target gene to be suppressed. Also, the expression of asuitable sense RNA can bring about a reduction of the expression ofendogenous genes, by means of what is known as co-suppression (EP-A1 0465 572). Moreover, further methods such as, for example, the regulationof gene expression by means of viral expression systems (virus-inducedgene silencing, VIGS; WO 98/36083, WO 99/15682) exist. Especiallypreferred is the expression of a double-stranded RNA for reducing thegene expression of a target gene. WO 99/32619 and WO 99/53050 describemethods for inhibiting individual target genes using an RNA withdouble-stranded structure, where the target gene and the region of theRNA duplex have at least partial identity (see also: Montgomery M K etal. (1998) Proc Natl Acad Sci USA 95:15502-15507; Sharp P A (1999) Genes& Development 13(2):139-141; Fire A et al. (1998) Nature 391:806-11).The method is currently also referred to as RNA interference (RNAi).

Preferred applications where the reduction (suppression) of geneexpression brings about an advantageous phenotype comprise by way ofexample, but not by limitation:

-   1. Modification of the carbohydrate composition    -   A modification of the carbohydrate composition can be achieved        for example by reducing the gene expression of genes of the        carbohydrate metabolism or of carbohydrate biosynthesis, for        example the biosynthesis of amylose, pectins, cellulose or        cell-wall carbohydrates. A multiplicity of cellular processes        (maturation, starch composition, starch content and the like)        can thus be influenced in an advantageous manner. Target genes        which may be mentioned by way of example, but not by limitation,        are phosphorylases, starch synthetases, branching enzymes,        sucrose-6-phosphate synthetases, sucrose-6-phosphate        phosphatases, lipoxygenases (Griffiths A. et al. (1999)        Postharvest Biology & Technology 17(3):163-173), ADP-glucose        pyrophosphorylases, branching enzymes, debranching enzymes, and        various amylases. The genes in question are described (Dunwell J        M (2000) J Exp Botany 51 Spec No: 487-96; Brar D S et al. (1996)        Biotech Genet Eng Rev 13:167-79; Kishore G M and Somerville C        R (1993) Curr Opin Biotech 4(2):152-8). Advantageous genes for        influencing the carbohydrate metabolism—in particular starch        biosynthesis are—described in WO 92/11375, WO 92/11376, U.S.        Pat. No. 5,365,016 and WO 95/07355.    -   In a further advantageous embodiment, a shift of the        amylose/amylopectin ratio in starch can be brought about by        suppression of the two isoforms of the branching enzyme which        are responsible for the α-1,6-glycosidic linkage. Such        procedures are described (for example by Schwall G P et        al. (2000) Nat Biotechnol 18(5):551-554). Nucleic acid sequences        such as that of the potato starch branching enzyme II (GenBank        Acc. No.: AR123356; U.S. Pat. No. 6,169,226) or its homologs        from other genera and species are preferably used for this        purpose.    -   Especially advantageous is the reduction of starch mobilization        and conversion into sugars at low temperatures (cold sweetening)        by means of reducing the expression of glucan phosphorylase        (systematic name: 1,4-α-D-glucan:phosphate        α-D-glucosyltransferase; U.S. Pat. No. 5,998,710).-   2. Delayed fruit maturation    -   Delayed fruit maturation or a modified maturation phenotype        (prolonged maturation, later senescence) can be achieved for        example by reducing the gene expression of genes selected from        the group consisting of polygalacturonases, pectin esterases,        β-(1,4)glucanases (cellulases), β-galactanases        (β-galactosidases), or genes of ethylene biosynthesis, such as        1-aminocyclopropane-1-carboxylate synthase, adenosylmethionine        hydrolase (SAMase), aminocyclopropane-1-carboxylate deaminase,        aminocyclopropane-1-carboxylate oxidase, genes of carotenoid        biosynthesis such as, for example, genes of pre-phytoene        biosynthesis or phytoene biosynthesis, for example phytoene        desaturases, and O-methyltransferases, acyl carrier protein        (ACP), elongation factor, auxin-induced gene, cysteine(thiol)        proteinases, starch phosphorylases, pyruvate decarboxylases,        chalcone reductases, protein kinases, auxin-related gene,        sucrose transporters, meristem pattern gene. Further        advantageous genes are described for example in WO 91/16440, WO        91/05865, WO 91/16426, WO 92/17596, WO 93/07275 or WO 92/04456.        Especially preferred is the reduction of the expression of        polygalacturonase for the prevention of cell degradation and        mushiness of plants and fruits, for example tomatoes. Nucleic        acid sequences such as that of the tomato polygalacturonase gene        (GenBank Acc. No.: x14074) or its homologs are preferably used        for this purpose.-   3. Improved protection against abiotic stress factors (heat, chill,    drought, elevated moisture, pollutants, UV radiation). It is    preferred to reduce the expression of genes which are implicated in    stress reactions.-   4. Reduction of the storage protein content    -   The reduction of the gene expression of genes encoding storage        proteins (hereinbelow SPs) has numerous advantages, such as, for        example, the reduction of the allergenic potential or        modification regarding composition or quantity of other        metabolites, such as, for example, oil or starch content.-   5. Obtaining a resistance to plant pathogens    -   Resistance to plant pathogens such as arachnids, fungi, insects,        nematodes, protozoans, viruses, bacteria and diseases can be        achieved by reducing the gene expression of genes which are        essential for the growth, survival, certain developmental stages        (for example pupation) or the multiplication of a specific        pathogen. Such a reduction can bring about a complete inhibition        of the abovementioned steps, or else a delay of same. They can        take the form of plant genes which for example make possible the        penetration of the pathogen, but may also be homologous pathogen        genes. The transgenically expressed nucleic acid sequence (for        example the double-stranded RNA) is preferably directed against        genes of the pathogen. The antipathogenic agent which acts may        be, in this context, the transgenically expressed nucleic acid        sequence itself (for example the double-stranded RNA), but also        the transgenic expression cassettes or transgenic organisms. The        plants themselves, in the form of a transgenic organism, may        contain the agents and pass them on to the pathogens, for        example in the form of a stomach poison. Various essential genes        of a variety of pathogens are known to the skilled worker (for        example for nematode resistance WO 93/10251, WO 94/17194).-   6. Most preferred as pathogens are fungal pathogens such as    Phytophthora infestans, Fusarium nivale, Fusarium graminearum,    Fusarium culmorum, Fusarium oxysporum, Blumeria graminis,    Magnaporthe grisea, Sclerotinia sclerotium, Septoria nodorum,    Septoria tritici, Alternaria brassicae, Phoma lingam, bacterial    pathogens such as Corynebacterium sepedonicum, Erwinia carotovora,    Erwinia amylovora, Streptomyces scabies, Pseudomonas syringae pv.    tabaci, Pseudomonas syringae pv. phaseolicola, Pseudomonas syringae    pv. tomato, Xanthomonas campestris pv. malvacearum and Xanthomonas    campestris pv. oryzae, and nematodes such as Globodera    rostochiensis, G. pallida, Heterodera schachtii, Heterodera avenae,    Ditylenchus dipsaci, Anguina tritici and Meloidogyne hapla.-   7. Virus resistance can be achieved for example by reducing the    expression of a viral coat protein, a viral replicase, a viral    protease and the like. A large number of plant viruses and suitable    target genes are known to the skilled worker.-   8. Reduction of undesired, allergenic or toxic plant constituents    such as, for example, glucosinolates or patatin. Suitable target    genes are described (in WO 97/16559, inter alia). The target genes    which are preferred for reduction of allergenic proteins are    described for example by Tada Y et al. (1996) FEBS Lett    391(3):341-345 or Nakamura R (1996) Biosci Biotechnol Biochem    60(8):1215-1221.-   9. Delayed signs of senescence. Suitable target genes are, inter    alia, cinnamoyl-CoA:NADPH reductases or cinnamoyl-alcohol    dehydrogenases. Further target genes are described (in WO 95/07993,    inter alia).-   10. Reduction of the susceptibility to bruising of, for example,    potatoes by reducing for example polyphenol oxidase (WO 94/03607)    and the like.-   11. Increase of the methionine content by reducing threonine    biosynthesis, for example by reducing the expression of threonine    synthase (Zeh M et al. (2001) Plant Physiol 127(3):792-802).

Antisense nucleic acid firstly means a nucleic acid sequence which isfully or in part complementary to at least part of the sense strand ofsaid target protein. The skilled worker knows that an alternative is theuse of the cDNA or the corresponding gene as starting template forsuitable antisense constructs. Preferably, the antisense nucleic acid iscomplementary to the coding region of the target protein or part ofsame. However, the antisense nucleic acid may also be complementary tothe noncoding region or part of same. Starting from the sequenceinformation of a target protein, an antisense nucleic acid can bedesigned in the manner with which the skilled worker is familiar, takinginto consideration the Watson-Crick base pair rules. An antisensenucleic acid can be complementary to all or part of the nucleic acidsequence of a target protein. In a preferred embodiment, the antisensenucleic acid is an oligonucleotide with a length of, for example, 25,30, 35, 40, 45 or 50 nucleotides.

The antisense strategy can advantageously be combined with a ribozymemethod. Ribozymes are catalytically active RNA sequences which, whenlinked with the antisense sequences, catalytically cleave the targetsequences (Tanner N K (1999) FEMS Microbiol Rev 23(3):257-75). Theefficiency of an antisense strategy may thereby be increased. Theexpression of ribozymes for reducing certain proteins is known to theskilled worker and described for example in EP-A1 0 291 533, EP-A1 0 321201 and EP A1 0 360 257. Suitable target sequences and ribozymes can beidentified for example as described by Steinecke (Ribozymes, Methods inCell Biology 50, Galbraith et al. eds Academic Press, Inc. (1995),449-460) by calculating the secondary structures of ribozyme and targetRNA and also by their interaction (Bayley C C et al. (1992) Plant MolBiol 18(2):353-361; Lloyd A M and Davis R W et al. (1994) Mol Gen Genet242(6):653-657). Examples which may be mentioned are hammerheadribozymes (Haselhoff and Gerlach (1988) Nature 334:585-591). Preferredribozymes are based on derivatives of the Tetrahymena L-19 IVS RNA (U.S.Pat. No. 4,987,071; U.S. Pat. No. 5,116,742). Further ribozymes withselectivity for an L119 mRNA can be selected (Bartel D and Szostak J W(1993) Science 261:1411-1418).

Also comprised is the use of the above-described sequences in senseorientation which, as the skilled worker will know, can lead toco-suppression. The expression of sense RNA to an endogenous gene canreduce or eliminate expression thereof, in a similar manner to what hasbeen described for antisense approaches (Goring et al. (1991) Proc NatlAcad Sci USA, 88:1770-1774; Smith et al. (1990) Mol Gen Genet224:447-481; Napoli et al. (1990) Plant Cell 2:279-289; Van der Krol etal. (1990) Plant Cell 2:291-299). In this context, the constructintroduced may represent the gene to be reduced either fully or only inpart. No possibility of translation is necessary.

Also very especially preferred is the use of methods such as generegulation by means of double-stranded RNA (double-stranded RNAinterference). Such methods are known to the skilled worker anddescribed in detail (for example Matzke M A et al. (2000) Plant Mol Biol43:401-415; Fire A. et al. (1998) Nature 391:806-811; WO 99/32619; WO99/53050; WO 00/68374; WO 00/44914; WO 00/44895; WO 00/49035; WO00/63364). The methods and processes described in the references citedare expressly referred to. Here, a highly efficient suppression ofnative genes is brought about by the simultaneous introduction of strandand counterstrand.

The transgenic expression cassettes according to the invention andtransgenic expression vectors derived from them may comprise furtherfunctional elements. The term functional element is to be understoodbroadly and means all those elements which have an effect on thegeneration, multiplication or function of the transgenic expressioncassettes according to the invention or on transgenic expression vectorsor organisms derived from them. The following may be mentioned by way ofexample, but not by limitation:

1. Selection Markers

The term “selection marker” comprises not only positive selectionmarkers, which confer a resistance to an antibiotic, herbicide or otherbiocide, but also negative selection markers, which confer a sensitivityto precisely the abovementioned, and also markers which confer a growthadvantage to the transformed organism (for example by expression of keygenes of cytokine biosynthesis; Ebinuma H et al. (2000) Proc Natl AcadSci USA 94:2117-2121). In the case of positive selection, only thoseorganisms which express the selection marker in question thrive, whileprecisely these organisms die in the case of negative selection. The useof a positive selection marker is preferred in the generation oftransgenic plants. Furthermore preferred is the use of selection markerswhich confer growth advantages. Negative selection markers can be usedadvantageously when the task at hand consists in eliminating certaingenes or genome segments from an organism (for example for the purposesof a hybridization process).

i) Positive Selection Markers:

The selectable marker introduced with the transgenic expression cassetteconfers resistance to a biocide, for example a herbicide (such asphosphinothricin, glyphosate or bromoxynil), a metabolic inhibitor (suchas 2-deoxyglucose-6-phosphate; WO 98/45456) or an antibiotic (such as,for example, tetracyclins, ampicillin, kanamycin, G 418, neomycin,bleomycin or hygromycin) to the successfully transformed cells. Theselection marker permits the selection of the transformed cells fromuntransformed cells (McCormick et al. (1986) Plant Cell Reports 5:81-84.

Especially preferred selection markers are those which confer resistanceto herbicides. Selection markers which may be mentioned by way ofexample are:

-   -   DNA sequences which encode phosphinothricin acetyltransferases        (PAT) (also referred to as Bialophos® resistance gene (bar)),        which acetylate the free amino group of the glutamine synthase        inhibitor phosphinothricin (PPT) and thus detoxify the PPT (de        Block et al. (1987) EMBO J. 6:2513-2518; Vikkers JE et        al. (1996) Plant Mol Biol Reporter 14:363-368; Thompson CJ et        al. (1987) EMBO J. 6:2519-2523). The bar/PAT gene can be        isolated for example from Streptomyces hygroscopicus or S.        viridochromogenes. Such sequences are known to the skilled        worker (Streptomyces hygroscopicus GenBank Acc. No.: X17220 and        X05822; Streptomyces viridochromogenes GenBank Acc. No.: M22827        and X65195; U.S. Pat. No. 5,489,520). Synthetic genes are        further described for expression in plastids (GenBank Acc. No.:        AJ028212).    -   5-Enolpyruvylshikimate-3-phosphate synthase genes (EPSP synthase        genes), which confer resistance to Glyphosat®        (N-(phosphonomethyl)glycin). The nonselective herbicide        glyphosate has 5-enolpyruvyl-3-phosphoshikimate synthase (EPSPS)        as molecular target. This enzyme has a key function in the        biosynthesis of aromatic amino acids in plants (Steinrucken H C        et al. (1980) Biochem Biophys Res Commun 94:1207-1212; Levin J G        and Sprinson D B (1964) J Biol Chem 239:1142-1150; Cole D        J (1985) Mode of action of glyphosate; A literature analysis,        pp. 48-74. In: Grossbard E and Atkinson D (eds.). The herbicide        glyphosate. Buttersworths, Boston). Glyphosate-tolerant EPSPS        variants are preferably used as selection markers (Padgette S R        et al. (1996). New weed control opportunities: development of        soybeans with a Roundup Ready gene. In: Herbicide Resistant        Crops (Duke S O, ed.), pp. 53-84. CRC Press, Boca Raton, Fla.;        Saroha M K and Malik V S (1998) J Plant Biochemistry and        Biotechnology 7:65-72). The EPSPS gene of Agrobacterium sp.        strain CP4 has a natural tolerance to glyphosate which can be        transferred to corresponding transgenic plants. The CP4 EPSPS        gene has been cloned from Agrobacterium sp. strain CP4 (Padgette        S R et al. (1995) Crop Science 35(5):1451-1461).        5-Enolpyruvylshikimate-3-phosphate synthases which are        glyphosate-tolerant, such as, for example, those described in        U.S. Pat. No. 5,510,471; U.S. Pat. No. 5,776,760; U.S. Pat. No.        5,864,425; U.S. Pat. No. 5,633,435; U.S. Pat. No. 5,627,061;        U.S. Pat. No. 5,463,175; EP 0 218 571, are preferred, the        sequences described in the patents in each case also being        deposited in GenBank. Further sequences are described under        GenBank Accession X63374. The aroA gene (GenBank Acc. No.:        M10947) is furthermore preferred.    -   the gox gene (glyphosate oxidoreductase), which encodes the        Glyphosat®-degrading enzymes. GOX (for example the glyphosate        oxidoreductase from Achromobacter sp.) catalyzes the cleavage of        a C—N bond in glyphosate, which is thus converted into        aminomethylphosphonic acid (AMPA) and glyoxylate. GOX can        thereby confer resistance to glyphosate (Padgette S R et        al. (1996) J Nutr 126(3):702-16; Shah D et al. (1986) Science        233:478-481).    -   the deh gene (encoding a dehalogenase which inactivates        Dalapon®; WO 99/27116; GenBank Acc. No.: AX022822, AX022820)    -   bxn genes, which encode Bromoxynil®-degrading nitrilase enzymes,        for example the Klebsiella ozanenae nitrilase. Sequences can be        found in GenBank for example under the Acc. No.: E01313 and        J03196.    -   Neomycin phosphotransferases (npt) confer resistance to        antibiotics (aminoglycosides) such as neomycin, G418,        hygromycin, paromomycin or kanamycin, by reducing their        inhibitory action by means of a phosphorylation reaction.        Especially preferred is the nptII gene (GenBank Acc. No.:        AF080390; AF080389). Moreover, the gene is already a component        in a large number of expression vectors and can be isolated from        them using methods with which the skilled worker is familiar        (such as, for example, polymerase chain reaction) (GenBank Acc.        No.: AF234316 pCAMBIA-2301; AF234315 pCAMBIA-2300, AF234314        pCAMBIA-2201). The NPTII gene encodes an aminoglycoside        3′-O-phosphotransferase from E. coli, Tn5 (GenBank Acc. No.:        U00004 position 1401-2300; Beck et al. (1982) Gene 19 327-336).    -   the DOG^(R)l gene was isolated from the yeast Saccharomyces        cerevisiae (EP 0 807 836) and it encodes a        2-deoxyglucose-6-phosphate phosphatase, which confers resistance        to 2-DOG (Randez-Gil et al. (1995) Yeast 11:1233-1240; Sanz et        al. (1994) Yeast 10:1195-1202; GenBank Acc. No.: NC001140        position 194799-194056).    -   Sulfonylurea- and imidazolinone-inactivating acetolactate        synthases, which confer resistance to imidazolinone/sulfonylurea        herbicides. Examples which may be mentioned of imidazolinone        herbicides are the active substances imazamethabenz-methyl,        imazzamox, imazapyr, imazaquin and imazethapyr. Examples of        sulfonylurea herbicides which may be mentioned are        amidosulforon, azimsulfuron, chlorimuronethyl, chlorsulfuron,        cinosulfuron, imazosulforon, oxasulforon, prosulforon,        rimsulforon, sulfosulforon. The skilled worker is familiar with        a large number of further active substances from the        abovementioned classes. The sequence for the Arabidopsis        thaliana Csr 1.2 gene (EC 4.1.3.18) which has been deposited        under the GenBank Acc. No.: X51514, is suitable for example        (Sathasivan K et al. (1990) Nucleic Acids Res. 18(8):2188).        Acetolactate synthases, which confer resistance to imidazolinone        herbicides, are furthermore described under the GenBank Acc.        Nos: AB049823, AF094326, X07645, X07644, A19547, A19546, A19545,        105376 (EP 0 257 993), 105.373 (EP 0 257 993), AL133315.    -   Hygromycin phosphotransferases (e.g. GenBank Acc. No.: X74325)        which confer resistance to the antibiotic hygromycin. The gene        is a component of a large number of expression vectors and can        be isolated from them using methods with which the skilled        worker is familiar (such as, for example, polymerase chain        reaction) (GenBank Acc. No.: AF294981 pINDEX4; AF234301        pCAMBIA-1380; AF234300 pCAMBIA-1304; AF234299 pCAMBIA-1303;        AF234298 pCAMBIA-1302; AF354046 pCAMBIA-1305; AF354045        pCAMBIA-1305.1)    -   genes for resistance to        -   a) chloramphenicol (chloramphenicol acetyltransferase),        -   b) tetracyclin; various resistance genes have been            described, for example GenBank Acc. No.: X65876, X51366.            Moreover, the gene is already a component of a large number            of expression vectors and can be isolated therefrom using            methods known to the skilled worker (such as, for example,            polymerase chain reaction)        -   c) Streptomycin; various resistance genes have been            described, for example with the GenBank Acc. No.: AJ278607.        -   d) Zeocin; the corresponding resistance gene is a component            of a large number of cloning vectors (for example GenBank            Acc. No.: L36849 cloning vector PZEO) and can be isolated            from these using methods known to the skilled worker (such            as, for example, polymerase chain reaction).        -   e) Ampicillin (β-lactamase gene; Datta N, Richmond            M H. (1966) Biochem J 98(1):204-9; Heffron F et            al. (1975) J. Bacteriol 122:250-256; the Amp gene was first            cloned for generating the E. coli vector pBR322; Bolivar F            et al. (1977) Gene 2:95-114). The sequence is a component of            a large number of cloning vectors and can be isolated from            them using methods known to the skilled worker (such as, for            example, polymerase chain reaction).    -   Genes such as the isopentenyl transferase from Agrobacterium        tumefaciens (strain:PO22) (GenBank Acc. No.: AB025109). The ipt        gene is a key enzyme of cytokine biosynthesis. Its        overexpression facilitates the regeneration of plants (for        example selection on cytokine-free medium). The method for        utilizing the ipt gene has been described (Ebinuma H et        al. (2000) Proc Natl Acad Sci USA 94:2117-2121; Ebinuma H et        al. (2000) Selection of marker-free transgenic plants using the        oncogenes (ipt, rol A, B, C) of Agrobacterium as selectable        markers, In Molecular Biology of Woody Plants. Kluwer Academic        Publishers).    -   Various other positive selection markers which confer a growth        advantage to the transformed plants over nontransformed plants,        and methods for their use, have been described, inter.alia, in        EP-A 0 601 092. Examples which may be mentioned are        β-glucuronidase (in conjunction with, for example, cytokinin        glucuronide), mannose-6-phosphate isomerase (in conjunction with        mannose), UDP-galactose 4-epimerase (in conjunction with, for        example, galactose), with mannose-6-phosphate isomerase in        conjunction with mannose being especially preferred.        ii) Negative Selection Markers

Negative selection markers make possible for example the selection oforganisms with successfully deleted sequences which comprise the markergene (Koprek T et al. (1999) The Plant Journal 19(6):719-726). Whencarrying out a negative selection, for example a compound whichotherwise has no disadvantageous effect on the plant is converted into acompound which is disadvantageous, for example owing to the negativeselection marker introduced into the plant. Genes which have adisadvantageous effect per se are furthermore suitable. Negativeselection markers which may be mentioned by way of example, but not bylimitation, are TK thymidine kinase (TK), diphtheria toxin A fragment(DT-A), the coda gene product encoding a cytosine deaminase (Gleave A Pet al. (1999) Plant Mol Biol 40(2):223-35; Perera R J et al. (1993)Plant Mol Biol 23(4): 793-799; Stougaard J (1993) Plant J 3:755-761),the cytochrome P450 gene (Koprek et al. (1999) Plant J 16:719-726),genes encoding a haloalkane dehalogenase (Naested H (1999) Plant J18:571-576), the iaaH gene (Sundaresan V et al. (1995) Genes &Development 9:1797-1810.) or the tms2 gene (Fedoroff N V & Smith D L(1993) Plant J 3:273-289).

2) Reporter Genes

Reporter genes encode readily quantifiable proteins which, via theircolor or enzyme activity, allow an assessment of the transformationefficiency, the site or time of expression (see also Schenbron E,Groskreutz D. Mol Biotechnol. 1999; 13(1):29-44). Examples which may bementioned are:

-   -   “green fluorescence protein” (GFP) (Chui W L et al. (1996), Curr        Biol 6:325-330; Leffel S M et al. (1997) Biotechniques        23(5):912-8; Sheen et al. (1995) Plant J 8(5):777-784; Haseloff        et al. (1997) Proc Natl Acad Sci USA 94(6):2122-2127; Reichel et        al. (1996) Proc Natl Acad Sci USA 93(12):5888-5893; Tian et        al. (1997) Plant Cell Rep 16:267-271; WO 97/41228).    -   Chloramphenicol transferase (Fromm et al. (1985) Proc Natl Acad        Sci USA 82:5824-5828),    -   Luciferase (Millar et al. (1992) Plant Mol Biol Rep 10:324-414;        Ow et al. (1986) Science 234:856-859); allows detection via        bioluminescence.    -   β-Galactosidase, encodes an enzyme for which a variety of        chromogenic substrates are available.    -   β-Glucuronidase (GUS) (Jefferson et al. (1987) EMBO J.        6:3901-3907) or the uidA gene, which encodes an enzyme for a        variety of chromogenic substrates.    -   R-Locus gene product: protein which regulates the production of        anthocyanine pigments (red coloration) in plant tissue and thus        makes possible the direct analysis of the promoter activity        without addition of further auxiliary substances or chromogenic        substrates (Dellaporta et al. (1988) In: Chromosome Structure        and Function: Impact of New Concepts, 18^(th) Stadler Genetics        Symposium, 11:263-282).    -   Tyrosinase (Katz et al. (1983) J Gen Microbiol 129:2703-2714),        an enzyme which oxidizes tyrosine to DOPA and dopaquinone, which        subsequently form melanin, which can be detected readily.    -   Aequorin (Prasher et al. (1985) Biochem Biophys Res Commun        126(3):1259-1268), can be used in the calcium-sensitive        bioluminescence detection.        3) Replication Origins

Replication origins ensure the multiplication of the transgenicexpression cassettes or transgenic expression vectors according to theinvention in, for example, E. coli or agrobacteria. Examples which maybe mentioned are OR1 (origin of DNA replication), the pBR322 ori or theP15A ori (Sambrook et al.: Molecular Cloning. A Laboratory Manual,2^(nd) ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989). Examples of replication origins which are functional inAgrobacterium are pRK2, pRi, PVS1 or pSA.

4) Border Sequences

“Border sequences” (such as, for example, the right or left border ofthe T-DNA) make possible an agrobacteria-mediated transfer into plantcells for the transfer and integration into the plant genome.

5) Multiple Cloning Regions (MCS) Permit and Facilitate the Insertion ofOne or More Nucleic Acid Sequences.

Also according to the invention are transgenic expression vectors whichcomprise the above-described transgenic expression cassettes. Vectorsgenerally means structures which are capable of replication and whichare preferably host-specific, and which make possible the uptake ofnucleic acid sequences and their transfer into other cells. Examples ofvectors can be plasmids, cosmids, phages, viruses or else agrobacteria.Vectors which are particularly suitable for the purposes of plantbiotechnology are described hereinbelow.

Another subject of the invention relates to transgenic organisms,transformed with at least one transgenic expression cassette accordingto the invention or one transgenic expression vector according to theinvention, and to cells, cell cultures, tissues, parts—such as, forexample in the case of plant organisms, leaves, roots and the like—orpropagation material derived from such organisms.

Organism, starting organisms or host organisms are understood as meaningprokaryotic or eukaryotic organisms such as, for example, microorganismsor plant organisms. Preferred microorganisms are bacteria, yeasts, algaeor fungi.

Preferred bacteria are bacteria of the genus Escherichia, Erwinia,Agrobacterium, Flavobacterium, Alcaligenes or cyanobacteria, for exampleof the genus Synechocystis.

Especially preferred are microorganisms which are capable of infectingplants and thus of transferring the cassettes according to theinvention. Preferred microorganisms are those from the genusAgrobacterium and in particular the species Agrobacterium tumefaciens.

Preferred yeasts are Candida, Saccharomyces, Hansenula or Pichia.

Preferred fungi are Aspergillus, Trichoderma, Ashbya, Neurospora,Fusarium, Beauveria or further fungi described in Indian Chem Engr.Section B. Vol 37, No 1,2 (1995) on page 15, Table 6.

Host or starting organisms which are preferred as transgenic organismsare, above all, plant organisms. Plant organisms generally means allthose organisms which are capable of photosynthesis.

Included as plant organisms within the scope of the invention are allgenera and species of the higher and lower plants of the plant kingdom.The mature plants, seeds, tubers, beets/swollen tap roots, fruits,shoots and seedlings and also parts, propagation material and cultures,for example cell cultures, derived therefrom are also included. Matureplants means plants at any developmental stage beyond the seedling.Seedling means a young immature plant in an early developmental stage.

Annual, perennial, monocotyledonous and dicotyledonous plants arepreferred host organisms for preparing transgenic plants. The expressionof genes is furthermore advantageous in all ornamental plants, useful orornamental trees, flowers, cut flowers, shrubs or lawns. Plants whichmay be mentioned by way of example but not by limitation areangiosperms, bryophytes such as, for example, Hepaticae (liverworts) andMusci (mosses); pteridophytes such as ferns, horsetail and club mosses;gymnosperms such as conifers, cycades, ginkgo and Gnetalae; algae suchas Chlorophyceae, Phaeophpyceae, Rhodophyceae, Myxophyceae,Xanthophyceae, Bacillariophyceae (diatoms) and Euglenophyceae.

Preference is given to plants of the following plant families:Amaranthaceae, Asteraceae, Brassicaceae, carophyllaceae, Chenopodiaceae,Compositae, Cruciferae, Cucurbitaceae, Labiatae, Leguminosae,Papilionoideae, Liliaceae, Linaceae, Malvaceae, Rosaceae, Rubiaceae,Saxifragaceae, Scrophulariaceae, Solanacea, Sterculiaceae,Tetragoniacea, Theaceae, Umbelliferae.

Preferred monocotyledonous plants are in particular selected from themonocotyledonous crop plants, for example of the Gramineae family, suchas rice, corn, wheat, or other cereal species such as barley, malt, rye,triticale or oats, and also sugar cane and all grass species.

Preferred dicotyledonous plants are in particular selected from thedicotyledonous crop plants, for example

-   -   Asteraceae such as sunflower, Tagetes or Calendula and others,    -   Compositae, particularly the genus Lactuca, very particularly        the species sativa (lettuce), and others,    -   Cruciferae, particularly the genus Brassica, very particularly        the species napus (oilseed rape), campestris (beet), oleracea cv        Tastie (cabbage), oleracea cv Snowball Y (cauliflower) and        oleracea cv Emperor (broccoli), and further cabbage species; and        the genus Arabidopsis, very particularly the species thaliana,        and also cress or canola, and others,    -   Cucurbitaceae such as melon, pumpkin or zucchini, and others,    -   Leguminosae particularly the genus Glycine, very particularly        the species max (soybean), soya and also alfalfa, pea, bean        plants or peanut, and others,    -   Rubiaceae, preferably of the subclass Lamiidae, such as, for        example, Coffea arabica or Coffea liberica (coffee bush), and        others,    -   Solanaceae, in particular the genus Lycopersicon, very        particularly the species esculentum (tomato), and the genus        Solanum, very particularly the species tuberosum (potato) and        melongena (aubergine) and the genus Capsicum, very especially        the species annum (pepper), and also tobacco, and others,    -   Sterculiaceae, preferably of the subclass Dilleniidae, such as,        for example, Theobroma cacao (cacao bush) and others,    -   Theaceae, preferably of the subclass Dilleniidae, such as, for        example, Camellia sinensis or Thea sinensis (tea shrub) and        others,    -   Umbelliferae, preferably the genus Daucus, very particularly the        species carota (carrot), and Apium, very particularly the        species graveolens dulce (celery), and others;    -   Chenopodiaceae, preferably the genus Beta vulgaris, in        particular the species Beta vulgaris ssp. vulgaris var.        altissima L. (sugar beet) and others;        and also linseed, cotton, hemp, flax, cucumber, spinach, carrot,        sugar beet and the various tree, nut and vine species, in        particular banana and kiwi fruit.

Plant organisms for the purposes of the invention are furthermore otherphotosynthetically active capable organisms, such as, for example,algae, and mosses. Preferred algae are green algae, such as, forexample, algae of the genus Haematococcus, Phaedactylum tricornatum,Volvox or Dunaliella.

Most preferred are plants of the family Solanaceae, especially the genusLycopersicon, very especially the species esculentum (tomato), the genusSolanum, very especially the species tuberosum (potato) and melongena(aubergine), of the family Chenopodiaceae, in particular the genus Betavulgaris, in particular the species Beta vulgaris ssp. vulgaris var.altissima L. (sugar beet) and others, of the family Leguminosae,especially the genus Glycine, very especially the species max (soybean)and alfalfa, pea, bean plants, especially the genus Vicia or peanut andothers, and other plants with starch-containing seeds, tubers,beets/swollen tap roots, fruits or tissues. Preferred among these, inturn, are tomato, potato, aubergine, soybean, alfalfa, pea, field bean,fodder beet, sugar beet and peanut.

The preparation of a transformed organism or of a transformed cellrequires introducing the appropriate DNA into the appropriate host cell.A multiplicity of methods is available for this process which isreferred to as transformation (see also Keown et al. 1990 Methods inEnzymology 185:527-537). Thus, by way of example, the DNA may beintroduced directly by microinjection or by bombardment with DNA-coatedmicroparticles. The cell may also be permeabilized chemically, forexample using polyethylene glycol, so that the DNA can enter the cellvia diffusion. The DNA may also be performed via protoplast fusion withother DNA-containing units such as minicells, cells, lysosomes orliposomes. Another suitable method for introducing DNA iselectroporation in which the cells are reversibly permeabilized by anelectric impulse.

In the case of plants, the methods described for transforming andregenerating plants from plant tissues or plant cells are utilized fortransient or stable transformation. Suitable methods are especiallyprotoplast transformation by polyethylene glycol-induced DNA uptake, thebiolistic method using the gene gun, the “particle bombardment” method,electroporation, the incubation of dry embryos in DNA-containingsolution and microinjection.

Apart from these “direct” transformation techniques, a transformationmay also be carried out by bacterial infection by means of Agrobacteriumtumefaciens or Agrobacterium rhizogenes. These strains contain a plasmid(Ti or Ri plasmid), a part of which (what is known as T-DNA) istransferred to the plant after infection with Agrobacterium andintegrated into the genome of the plant cell. The Agrobacterium-mediatedtransformation is best suited to dicotyledonous plant cells, whereas thedirect transformation techniques are suitable for any cell type.

A transgenic expression cassette of the invention may be introducedadvantageously into cells, preferably into plant cells, by usingvectors.

In an advantageous embodiment, the transgenic expression cassette isintroduced by means of plasmid vectors. Preference is given to thosetransgenic expression vectors which enable a stable integration of thetransgenic expression cassette into the host genome. In this context,host genome means the entire hereditary information of the host andcomprises for example not only the chromosomal DNA of the nucleus, butalso the DNA of the plastids and mitochondria. However, the insertioninto the chromosomal DNA of the nucleus is preferred.

In the case of injection or electroporation of DNA into plant cells, noparticular demands on the plasmid used are made. It is possible to usesimple plasmids such as those of the pUC series. If complete plants areto be regenerated from the transformed cells, it is necessary for anadditional selectable marker gene to be present on the plasmid.

Transformation techniques have been described for variousmonocotyledonous and dicotyledonous plant organisms. Furthermore,various possible plasmid vectors which normally contain a replicationorigin for propagation in E. coli and a marker gene for selection oftransformed bacteria are available for introducing foreign genes intoplants. Examples are pBR322, pUC series, M13 mp series, pACYC184 etc.

The transgenic expression cassette may be introduced into the vector viaa suitable restriction cleavage site. The resultant plasmid is firstintroduced into E. coli. Correctly transformed E. coli cells areselected, cultivated and the recombinant plasmid is obtained usingmethods familiar to the skilled worker. Restriction analysis andsequencing may be used in order to check the cloning step.

Transformed cells, i.e. those which contain the introduced DNAintegrated into the DNA of the host cell may be selected fromuntransformed cells, if a selectable marker is part of the introducedDNA. A marker may be, by way of example, any gene which is capable ofimparting a resistance to antibiotics or herbicides. Transformed cellswhich express such a marker gene are capable of surviving in thepresence of concentrations of an appropriate antibiotic or herbicide,which kill an untransformed wild type. Examples are the bar gene whichimparts resistance to the herbicide phosphinothricin (Rathore K S etal., Plant Mol Biol. 1993 March; 21(5):871-884), the nptII gene whichimparts resistance to kanamycin, the hpt gene which imparts resistanceto hygromycin and the EPSP gene which imparts resistance to theherbicide glyphosate.

Depending on the method of DNA introduction, further genes may berequired on the vector plasmid. If agrobacteria are used, the transgenicexpression cassette is to be integrated into specific plasmids, eitherinto an intermediate vector (shuttle vector) or a binary vector. If, forexample, a Ti or Ri plasmid is to be used for transformation, at leastthe right border, in most cases, however, the right and the left border,of the Ti or Ri plasmid T-DNA is connected as flanking region with thetransgenic expression cassette to be introduced. Preference is given tousing binary vectors. Binary vectors can replicate both in E. coli andin Agrobacterium. They normally contain a selection marker gene and alinker or polylinker flanked by the right and left T-DNA bordersequences. They may be transformed directly into Agrobacterium (Holsterset al., Mol. Gen. Genet. 163 (1978), 181-187). The selection marker genepermits selection of transformed agrobacteria; an example is the nptIIgene which imparts a resistance to kanamycin. The Agrobacterium which inthis case acts as the host organism should already contain a plasmidwith the vir region. This region is required for the transfer of T-DNAonto the plant cell. An Agrobacterium transformed in this way may beused for transformation of plant cells.

The use of T-DNA for transformation of plant cells has been intenselystudied and described (B. Jenes et al., Techniques for Gene Transfer,in: Transgenic Plants, Vol. 1, Engineering and Utilization, edited byKung S D and Wu R, Academic Press (1993), pp. 128-143 and in Potrykus(1991) Annu Rev Plant Physiol Plant Molec Biol 42:205-225; EP 120516;Hoekema, In: The Binary Plant Vector System, Offsetdrukkerij Kanters B.V., Alblasserdam, Chapter V; Fraley et al., Crit. Rev. Plant. Sci.,4:1-46 and An et al. (1985) EMBO J. 4:277-287). Various binary vectorsare known and partly commercially available, such as, for example,pBIN19 (Bevan et al. (1984) Nucl Acids Res 12:8711f.; ClontechLaboratories, Inc. USA) or pSUN derivatives (SunGene GmbH & Co. KGaA; WO02/00900). The expression cassette according to the invention can beinserted into these binary vectors and integrated into the plant genomeas described hereinbelow.

The DNA is transferred into the plant cell by coculturing plant explantswith Agrobacterium tumefaciens or Agrobacterium rhizogenes. Startingfrom infected plant material (e.g. leaf, root or stem parts, but alsoprotoplasts or plant cell suspensions), it is possible to regeneratewhole plants by using a suitable medium which may contain, for example,antibiotics or biocides for selection of transformed cells. The plantsobtained may then be screened for the presence of the introduced DNA, inthis case the transgenic expression cassette of the invention. As soonas the DNA has integrated into the host genome, the correspondinggenotype is normally stable and the corresponding insertion is alsofound again in subsequent generations. Normally, the integratedtransgenic expression cassette contains a selection marker which impartsto the transformed plant a resistance to a biocide (for example aherbicide), a metabolism inhibitor such as 2-DOG or an antibiotic suchas kanamycin, G 418, bleomycin, hygromycin or phosphinothricin etc. Theselection marker allows the selection of transformed cells fromuntransformed cells (McCormick et al. (1986) Plant Cell Reports5:81-84). The plants obtained may be cultivated and crossed in thecommon manner. Two or more generations should be cultured in order toensure that the genomic integration is stable and heritable.

As soon as a transformed plant cell has been prepared, it is possible toobtain a complete plant by using methods known to the skilled worker. Tothis end, callus cultures are used as starting point, by way of example.From these still undifferentiated cell masses, it is possible to induceformation of shoot and root in the known manner. The shoots obtained canbe planted out and cultivated.

The integration of the T-DNA can be determined e.g. on the basis of theefficacy of expression of the nucleic acids to be expressedtransgenically or of the selection marker for example in vitro by shootmeristem propagation using one of the above-described selection methods.

The invention further relates to cells, cell cultures, parts, such as,for example, roots, leaves, etc. in the case of transgenic plantorganisms, and transgenic propagation material such as seeds, tubers,beets/swollen tap roots or fruits derived from the above-describedtransgenic organisms.

Genetically modified plants of the invention, which can be consumed byhumans and animals, may also be used, for example directly or afterpreparation known per se, as foodstuffs or feedstuffs.

The invention further relates to the use of the above-describedtransgenic organisms of the invention and of the cells, cell cultures,parts, such as, for example, roots, leaves, etc., in the case oftransgenic plant organisms, and transgenic propagation material such asseeds, tubers, beets/swollen tap roots or fruits derived from them forthe production of food- or feedstuffs, pharmaceuticals or finechemicals.

Preference is further given to a method for the recombinant productionof pharmaceuticals or fine chemicals in host organisms, in which a hostorganism is transformed with one of the above-described transgenicexpression cassettes or transgenic expression vectors and saidtransgenic expression cassette contains one or more structural geneswhich code for the fine chemical of interest or catalyze thebiosynthesis of the fine chemical of interest, and the transformed hostorganism is cultivated and the fine chemical of interest is isolatedfrom the cultivation medium. This method is broadly applicable for finechemicals such as enzymes, vitamins, amino acids, sugars, fatty acids,natural and synthetic flavorings, aromatizing substances and colorants.Particular preference is given to the production of tocopherols andtocotrienols and also carotenoids. Cultivation of the transformed hostorganisms and isolation from said host organisms or from the cultivationmedium are carried out by means of the methods known to the skilledworker. The production of pharmaceuticals such as, for example,antibodies or vaccines is described in Hood E E, Jilka J M (1999) CurrOpin Biotechnol 10(4):382-6; Ma J K, Vine N D (1999) Curr Top MicrobiolImmunol 236:275-92.

Sequences

1. SEQ ID NO: 1 Promoter of the Vicia faba Pho1 gene 2. SEQ ID NO: 2Promoter and 5′-untranslated region of the Vicia faba Pho1 gene 3. SEQID NO: 3 Promoter and 5′-untranslated region of the Vicia faba Pho1 geneand sequence encoding the transit peptide of the Vicia faba Pho1 protein4. SEQ ID NO: 4 Oligonucleotide primer GP15′-GATTGTCTCTAGATGTAGGTGTGTTT-3′ 5. SEQ ID NO: 5 Oligonucleotide primerGP2 5′-CATGGAAGCCATggTTGAATTTCT-3′ 6. SEQ ID NO: 6 Oligonucleotideprimer GPSP 5′-TTCCTGATCCaTGgCTTTCTGTTTCGC-3′ 7. SEQ ID NO: 7 Nucleicacid sequence encoding the transit peptide of the Vicia faba plastidic1,4-α-D-glucan:phosphate α-D-glucosyltransferase 8. SEQ ID NO: 8 Aminoacid sequence encoding the transit peptide of the Vicia faba plastidic1,4-α-D-glucan:phosphate α-D-glucosyltransferase

DESCRIPTION OF THE FIGURES

FIG. 1: Analysis of tubers of potato plants transformed withPTGPGUS-kan. What is shown is the staining of X-Gluc-incubated disksfrom tubers of two independent lines (A and B; the dark colorcorresponds to the blue staining).

FIG. 2: Analysis of fruits of tomato plants transformed withPTGPGUS-kan. What is shown are fruits of a transgenic line (gp7) incomparison with wild-type fruits. The dark color of the transgenicfruits corresponds to the GUS blue staining. The dark color of thewild-type fruits corresponds to the natural red coloration of thefruits; here, no blue staining can be found. 1: green, immature fruit;2: orange-colored fruit; 3: red, mature fruit; 4: overripe fruit. It canbe seen that the blue staining decreases as the degree of maturation ofthe fruit increases.

FIG. 3: Analysis of fruits of tomato plants transformed withPTUSPGUS-kan. A: immature fruit; B: mature fruit. In contrast with thepromoters according to the invention, the USP promoter is only active inthe seed kernels.

FIG. 4: Starch staining of tomato disks in different develop entalstages. Top row (A) unstained; bottom row (B) starch staining withLugol's solution. It can be seen that the staining decreases with anincreasing degree of maturity of the fruit.

FIG. 5: Analysis of fruits of tomato plants transformed withPTGPGUS-kan. What is shown are the GUS expression activities in tissuesfrom two transgenic lines (7 and 23). Wild-type fruits showed in eachcase only one background level. A: leaf; B: root; C: skin, green; D:skin, orange; E: skin, red; F: flesh, green; G: flesh, orange; H: flesh,red; I: seeds, green; J: seeds, orange; K: seeds, red. A high activityis discernible in particular in the immature flesh and skin of tomatoes(Y axis: GUS activity measured in pM MU/min mg protein; MU:methylumbelliferone). It can be seen that the GUS activity decreaseswith increasing degree of maturity of the fruit.

EXAMPLES General Methods

Recombinant DNA techniques were carried out as described by Maniatis etal., Molecular Cloning—A Laboratory Manual (Cold Spring Harbor Lab.,Cold Spring Harbor, N.Y., 1982). The enzymes employed were used asspecified. The cloning vectors used were pUC18 (Yanisch-Perron C et al.(1985) Gene 33103-119), pBK-CMV (Stratagene) and pGUS1 (Pelemann J etal. (1989) Plant Cell 1:81-93). The vectors pGPTV-BAR and pGPTV-kan(Becker D et al. (1992) Plant Mol Biol 20:1195-1197) were employed forthe transformation of plants. Strain DH5a (Hanahan D (1983) J Mol Biol166:557-580) was used for the transformation into E. coli. Theagrobacteria strains EHA 105, GV3101 [pMP90], C58C1 [pGV2260] andLBA4404 were transformed directly by means of the freeze-thaw method asdescribed by An G (1987) Mol Gen Genet 207:210-216.

Example 1 Cloning the Promoter of the Vicia faba Glucan Phosphorylase

To isolate the promoter, genomic DNA of Vicia faba was cleaved withBlgII and ligated into the BamHI-cut ZAP Express Vector System (#239212)from Stratagene, and the phages were plated. A genomic Vicia faba DNAlibrary (in pBKCMV (Stratagene)) was screened with a sample of the cDNAclone Pho1 (GenBank Acc. No.: Z36880) and the genomic clone pBKVfGP22was isolated. Starting from the genomic clone pBKVfGP22,

-   i) the 5′-flanking region including the 5′-untranslated region and    the ATG start codon of the Pho1 gene and-   ii) the 5′-flanking region of the Pho1 gene including the putative    transit peptide (Pho1-TP) were amplified.-   a) PCR amplification of the Pho1 glucan phosphorylase promoter

Primer GP1 (SEQ ID NO: 4): 5′-GATTGTCTCTAGATGTAGGTGTGTTT-3′ Primer GP2(SEQ ID NO: 5): 5′-CATGGAAGCCATggTTGAATTTCT-3′

-   -   The ATG start codon (reverse complement) is shown in primer GP2        in bold. By replacing two T by g (in lower case letters), the        recognition site for the restriction enzyme NcoI Ort was        introduced directly at the start codon.        Reaction Mix:

2 μl pBKVfGP22 (1:500) 10 μl Ampli Taq buffer 0.5 μl Ampli Taqpolymerase 2 μl dNTP (10 mM) 2 μl GP1 (10 μM) 2 μl GP2 (10 μM) 81 μl H₂OPCR Conditions:

1 cycle: 5 minutes at 96° C. 25 cycles: 0.5 minutes at 48° C.; 1 minuteat 72° C.; 0.5 minutes at 96° C. 1 cycle: 0.5 minutes at 48° C.; 10minutes at 72° C.

-   b) PCR amplification of the glucan phosphorylase promoter including    the transit peptide (Pho1-TP)    -   According to Buchner et al. (Planta 199:64-73, 1996), a sequence        encoding a plastidic transit peptide is located at the N        terminus of the transcript. This sequence shows no significant        homologies with other chloroplastidic or amyloplastidic transit        peptides. According to Gavel's and Heijne's rule (FEBS Lett        261:455-458, 1990), the sequence corresponds with the consensus        of transit peptides. Accordingly, the transit peptide has a        length of 64 amino acids. The primer GPSP was chosen in such a        way that the environment of the cleavage site is retained owing        to 10 additional amino acids, thus ensuring largely reliable        processing of the transit peptides. The fragment was amplified        with the primers GP1 (see hereinabove; SEQ ID NO: 4) and GPSP        (SEQ ID NO: 6).

Primer GPSP (SEQ ID NO: 6): 5′-TTCCTGATCCaTGgCTTTCTGTTTCGC-3′

-   -   By replacing a T by a and A by g (lower case letters), the        recognition site of the restriction enzyme NcoI was introduced.        Reaction Mix:

2 μl pBKVfGP22 (1:500) 10 μl Ampli Taq buffer 0.5 μl Ampli Taqpolymerase 2 μl dNTP (10 mM) 2 μl GP1 (10 μM) 2 μl GPSP (10 μM) 81 μlH₂OPCR Conditions:

1 cycle: 5 minutes at 96° C. 25 cycles: 0.5 minutes at 48° C.; 1 minuteat 72° C.; 0.5 minutes at 96° C. 1 cycle: 0.5 minutes at 48° C.; 10minutes at 72° C.

The PCR products were purified and cloned into the vector pUC18 whichhad been cleaved with the restriction enzyme SmaI. The sequence of theresulting plasmids pGP and pGPSP2 was verified by sequence analysis.

Example 2 Construction of the Transgenic Expression Cassettes

a) Construction of the Pho1-Promoter-GUS Expression Cassette

The plasmid pGP was cleaved with the restriction enzymes PstI and NcoI.For the fusion with the GUS gene, the approx. 1.4 kb PstI/NcoI promoterfragment was cloned into the plasmid pGUS1 which had likewise beencleaved with PstI and NcoI, and the positive recombinants wereidentified by means of HindIII cleavage and sequence analysis. Theresulting plasmid was named pGPGUS.

b) Construction of the Pho1-TP-Promoter-GUS Expression Cassette

The plasmid pGPSP2 was cleaved with the restriction enzymes PstI andNcoI. For the fusion with the GUS gene, the approx. 1.6 kb PstI/NcoIpromoter fragment was cloned into the plasmid pGUS1 which had likewisebeen cleaved with PstI and NcoI, and the positive recombinants wereidentified by means of HindIII cleavage and sequence analysis. Theresulting plasmid was named pGPSPGUS.

c) Cloning the Pho1-Promoter-GUS Expression Cassette into the BinaryVector pPTV-Bar

The binary vector pGPTV-bar was cleaved with EcoRI and SmaI, madeblunt-ended with Klenow enzyme and religated. The resulting plasmidpPTV-bar served as binary vector for the following constructions. Theplasmid pGPGUS was cleaved with XbaI, and the fragment comprising theglucan phosphorylase promoter and the GUS gene was ligated into theXbaI-cut vector pPTV-bar. The resulting transgenic expression vector(plasmid) was named PTGPGUS.

d) Cloning the Pho1-TP-Promoter-GUS Expression Cassette into the BinaryVector pPTV-Bar

The plasmid PGPSPGUS was cleaved with XbaI, and the fragment comprisingthe glucan phosphorylase promoter, the transit peptide and the GUS genewas ligated into the XbaI-cut vector pPTV-bar. The resulting transgenicexpression vector (plasmid) was named PTGPSPGUS.

e) Preparation of the Plasmid PTGPGUSKan

To carry out the transformation into potato and tomato, the plasmidPTGPGUSKan was prepared. To this end, the binary vector pGPTV-kan wascleaved with EcoRI and SalI, and the EcoRI/SalI-cut fragment of theplasmid pGPGUS was cloned therein.

Example 3 Transformation of Tobacco, Oilseed Rape, Arabidopsis, Potatoand Tomato

a) Tobacco

For the transformation of tobacco plants (Nicotiana tabacum L. cv.Samsun N N), 10 ml of an overnight culture of Agrobacterium tumefaciensEHA105, transformed with the transgenic expression vectors PTGPGUS andPTGPSPGUS, which culture had grown under selection conditions, were spundown, the supernatant was discarded, and the bacteria were resuspendedin an equal volume of antibiotic-free medium. Leaf disks of sterileplants (diameter approx. 1 cm) were bathed in this bacterial solution ina sterile Petri dish. Thereafter, the leaf disks were plated in Petridishes on MS medium (Murashige and Skoog (1962) Physiol Plant 15:473ff.)supplemented with 2% sucrose and 0.8% Bacto agar. Following incubationfor two days in the dark at 25° C., they were transferred to MS mediumsupplemented with 100 mg/l kanamycin, 500 mg/l Claforan, 1 mg/lbenzylaminopurine (BAP), 0.2 mg/l naphthylacetic acid (NAA), 1.6%glucose and 0.8% Bacto agar, and culturing was continued (16 hourslight/8 hours dark). Growing shoots were transferred to hormone-free MSmedium supplemented with 2% sucrose, 250 mg/l Claforan and 0.8% Bactoagar.

b) Oilseed Rape

Oilseed rape was transformed by means of the petiole transformation ofMoloney et al. (Moloney MM et al. (1989) Plant Cell Reports 8:238-242).The Agrobacterium strain EHA105, transformed with the transgenicexpression vector PTGPSPGUS, was employed for the generation oftransgenic oilseed rape plants.

c) Arabidopsis

Arabidopsis was transformed by means of the floral dip method (Clough SJ and Bent A F (1998) Plant J 16:735-743). The Agrobacterium strainEHA105, transformed with the transgenic expression vector PTGPSPGUS wasemployed for the generation of transgenic Arabidopsis plants.

d) Potato

For the transformation of potato (Solanum tuberosum), leaf disks ofin-vitro plants were infected with Agrobacterium tumefaciens C58C1[pGV2260], transformed with the transgenic expression vector PTGPGUSKan,in liquid Murashige Skoog medium for 20 minutes and subsequentlycocultured for 2 days in the dark. After the coculture, the explantswere cultured on solid MS medium which comprises 1.6% glucose instead ofsucrose (MG) and which has been supplemented with 5 mg/l NAA, 0.1 mg/lBAP, 250 mg/l Timentin and 30 to 40 mg/l kanamycin (KIM) at 21° C. in alight/dark rhythm of 16 h/8 h. After this callus phase, the explantswere placed on shoot induction medium (SIM). SIM had the followingcomposition: MG supplemented with 2 mg/l zeatin riboside, 0.02 mg/l NAA,0.02 mg/l GA3, 250 mg/l Timentin, 30 to 40 mg/l kanamycin. Every twoweeks, the explants were transferred to fresh SIM. The shoots whichformed were rooted on MS medium supplemented with 2% sucrose and 250mg/l Timentin and 30 to 40 mg/l kanamycin.

e) Tomato

The starting explants for the transformation were cotyledons of seven-to ten-day-old seedlings of the line Microtom. The culture medium usedfor the germination is the medium of Murashige and Skoog (1962:Murashige and Skoog, 1962, Physiol Plant 15:473-) supplemented with 2%sucrose, pH 6.1. Germination takes place at 21° C. at a low light level(20-100 μE). After seven to ten days, the cotyledons are dividedtransversely and placed on the medium MSBN (MS, pH 6.1, 3% sucrose+1mg/l BAP, 0.1 mg/l NAA), which has been charged on the previous day withtobacco cells grown in suspension culture. The tobacco cells are coveredwith sterile filter paper without leaving air bubbles. The preculture ofthe explants on the above-described medium is carried out for three tofive days. Thereafter, the explants are infected with the Agrobacteriumtumefaciens strain LBA4404, which bears the binary plasmid with the geneto be transformed, in the following manner: the strain which had beencultured overnight in YEB medium with the antibiotic for the binaryplasmid at 28° C. is centrifuged. The bacterial pellet is resuspended inliquid MS medium (3% sucrose, pH 6.1) and brought to an optical densityof 0.3 (at 600 nm). The precultured explants are suspended and incubatedfor 30 minutes at room temperature with gentle agitation. Thereafter,the explants are dried using sterile filter paper and returned to theirpreculture medium for three days of coculturing (21° C.).

After coculturing, the explants are transferred to MSZ2 medium (MS pH6.1 supplemented with 3% sucrose, 2 mg/l zeatin, 100 mg/l kanamycin, 160mg/l Timentin) and retained for the selective regeneration at 21° C.under low-light conditions (20-100 μE, photoperiod 16 h/8 h). Theexplants are transferred every two to three weeks until shoots form.Small shoots can be excised from the explant and rooted on MS (pH 6.1+3%sucrose) 160 mg/l Timentin, 30 mg/l kanamycin, 0.1 mg/l IAA. Rootedplants are transferred into the greenhouse.

Example 4 Isolation of Genomic DNA

The genomic DNA of transgenic tobacco, Arabidopsis and oilseed rapeplants was isolated with the aid of the DNA isolation kit from Macherey& Nagel. In a first step, the transgenic lines were identified via PCR,using gene-specific primers. The integration of the foreign DNA wasanalyzed by means of Southern blot analyses of 201 g of DNA after asuitable restriction cleavage.

Example 5 Transient Expression Analysis of the Glucan PhosphorylasePromoter-GUS Cassettes

A transient promoter analysis was carried out to assay the activity ofthe glucan phosphorylase promoter in different tissues and plants. Thiswas done using the particle gun “Biolistic PDS-1000 System (BioRadLaboratories, Hercules, Calif.) with a vacuum of 27 mmHg. Themicrocarriers used were gold particles with a diameter of 1.0 μm, whichwere coated with plasmid DNA as specified by BioRad. To this end, 25 μlof a gold suspension (50 mg/ml) were mixed with 10 μl of Qiagen-purifiedplasmid DNA (1 μg/μl), 25 μl CaCl₂ (2.5 M) and 10 μl spermidine (0.1 M),left to stand briefly and then spun down. The coated gold particles werewashed with 70% and 100% ethanol. Embryos were bombarded with 2000 PSI,fruits of tomatoes and tubers of potatoes with 1800 PSI. The bombardedtissues were cultured for 36 hours in liquid medium and then treatedovernight at 37° C. with an X-Gluc solution. The blue spots werecounted.

After the bombardment with the plasmids pGPGUS and PGPSPGUS, no, orbarely visible, spots were found in the embryos of oilseed rape,sunflower, linseed, Vicia faba and soybean. The bombardment of disks ofthe potato tuber and fruit tissue of tomatoes resulted in markedly morespots; no substantial differences between the plasmids were observed. Aplasmid which comprises the GUS gene under the control of the USP(unknown seed protein; Baumlein et al. (1991) Mol Gen Genet 225:459-467)was used as control. Although the promoter brings about a seed-specificexpression, a multiplicity of blue spots were also observed in othersink tissues.

Example 6 Detection of the Tissue-Specific Expression

To determine the characteristics of the promoter, it is necessary toplace the promoter before what is known as a reporter gene, which makespossible a determination of the expression activity. An example whichmay be mentioned is the bacterial β-glucuronidase (Jefferson et al.(1987) EMBO J. 6:3901-3907). The β-glucuronidase activity can bedetermined in planta by means of a chromogenic substrate such as5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid (X-Gluc) in connectionwith an activity staining. To examine the tissue specificity, the planttissue is prepared and stained. It is particularly advantageous toexcise embryos from immature or mature seeds before staining them.Likewise, unopened flowers can be stained to detect a promoter activityin the pollen.

A second assay permits a quantitative determination of the GUS activityin the test tissue. β-Glucuronidase MUG(4-methylumbelliferyl-β-D-glucuronide) is used as substrate for thequantitative activity determination; the former is cleaved into MU(methylumbelliferone) and glucuronic acid.

High-level expression of the glucan phosphorylase promoter was detectedin the tubers of the analyzed potato plants which had been transformedwith the plasmid PTGPGUS-kan. FIG. 1 shows the GUS activity in disks oftubers from two lines. Apart from 2 lines, where there was barelydiscernible expression in the leaves, nothing was found that suggestedexpression in the leaves. Again, the correlation of the expression instarch-containing tissues is shown.

High-level glucuronidase expression was detected in the fruits of thetransgenic tomato plants which had been transformed with the plasmidPTGPGUS-kan. FIG. 2 shows the high-level expression during thedevelopment of the tomato fruit. In contrast, the PTUSPGUSkan plants,which had been generated for comparison purposes, showed no furtheractivities of this promoter in the fruits of the transgenic tomatoplants, besides a high level of seed-specific activity (FIG. 3).

No substantial activity of the glucan phosphorylase promoter wasdetected in tobacco, oilseed rape and Arabidopsis.

Example 7 Correlation of Starch Content and Expression Pattern of theGlucan Phosphorylase Promoter in Tomato Fruits

To demonstrate the correlation between starch-containing tissue andexpression of the glucan phosphorylase promoter, tomato disks werestained with Lugol's solution. Intense blue staining shows thedistribution of starch. As can be seen from FIG. 5, it is precisely inthe young tomato fruits that a large amount of starch is detected. Thedistribution pattern also agrees with the GUS expression pattern of thetransgenic fruits.

1. A method for directing transgenic expression of a nucleic acidsequence in carbohydrate-storing sink tissues of plants, which comprisesthe following steps: I. introducing, into plant cells, a transgenicexpression cassette, wherein the transgenic expression cassettecomprises at least the following elements: a) the promoter sequence ofthe gene encoding the Vicia faba plastidic 1,4-α-D-glucan:phosphateα-D-glucosyltransferase, or a fragment thereof having the same promoteractivity, and b) at least one further nucleic acid sequence, wherein thepromoter sequence or the fragment thereof and the at least one furthernucleic acid sequence are functionally linked together, and the furthernucleic acid sequence is heterologous in relation to the promotersequence, II. selecting transgenic cells which comprise said expressioncassette stably integrated into the genome, and III. regenerating intactplants from said transgenic cells, wherein the promoter sequence or thefragment thereof directs expression of the further nucleic acid sequencein carbohydrate-storing sink tissue, but essentially not in sourcetissues.
 2. The method according to claim 1, wherein the promotersequence comprises i) the nucleotide sequence of SEQ ID NO: 1, or ii) afragment of SEQ ID NO: 1 which directs expression of a nucleic acidsequence in carbohydrate-storing sink tissues of plants.
 3. An isolatednucleic acid sequence comprising: i) the promoter sequence of the geneof the Vicia faba plastidic 1,4-α-D -glucan:phosphateα-D-glucosyltransferase of SEQ ID NO: 1, or ii) a fragment of SEQ ID NO:1 which directs expression of a nucleic acid sequence incarbohydrate-storing sink tissues of plants.
 4. The isolated nucleicacid sequence according to claim 3, further comprising a nucleotidesequence encoding a transit peptide located in 3′ orientation to thepromoter sequence or the fragment thereof.
 5. The isolated nucleic acidsequence according to claim 4, wherein the nucleotide sequence encodinga transit peptide is the sequence of SEQ ID NO:
 8. 6. The isolatednucleic acid sequence according to claim 3, wherein the nucleic acidsequence is the sequence of SEQ ID NO: 2 or
 3. 7. A transgenicexpression cassette for the expression of a nucleic acid comprising: a)the promoter sequence of the gene encoding the Vicia faba plastidic1,4-α-D-glucan:phosphate α-D-glucosyltransferase, or a fragment thereofhaving the same promoter activity, and b) at least one further nucleicacid sequence, wherein the promoter sequence or the fragment thereof andthe at least one further nucleic acid sequence are functionally linkedtogether, and the further nucleic acid sequence is heterologous inrelation to the promoter sequence or the fragment thereof; and whereinthe promoter sequence or the fragment thereof directs expression of thefurther nucleic acid sequence in carbohydrate-storing sink tissue, butessentially not in source tissues.
 8. The transgenic expression cassetteaccording to claim 7, wherein the promoter sequence comprises i) thenucleotide sequence of SEQ ID NO: 1, or ii) a fragment of SEQ ID NO: 1which directs expression of a nucleic acid sequence incarbohydrate-storing sink tissues of plants.
 9. The transgenicexpression cassette according to claim 8, where the promoter sequence isthe sequence of SEQ ID NO: 2 or
 3. 10. The transgenic expressioncassette according to claim 7, wherein the at least one further nucleicacid sequence a) encodes a protein, or b) transcribes a sense RNA,antisense RNA or double-stranded RNA.
 11. A transgenic expression vectorcomprising the nucleic acid sequence according to claim
 3. 12. Atransgenic organism transformed with the trausgenic expression cassetteaccording to claim
 7. 13. The transgenic organism according to claim 12,selected from the group consisting of bacteria, yeasts, fungi, nonhumananimal organisms and plant organisms.
 14. The transgenic organismaccording to claim 12, selected from the group consisting of tomato,potato, aubergine, soybean, alfalfa, pea, field bean, fodder beet, sugarbeet and peanut.
 15. A cell culture, part, organ, tissue or transgenicpropagation material derived from the transgenic organism according toclaim
 12. 16. A method for the transgenic expression of a nucleic acidcomprising growing or culturing the transgenic organism according toclaim 12 or cell cultures, parts, organs, tissues or transgenicpropagation material derived therefrom.
 17. A method for the productionof foodstuffs, feedstuffs, seed, pharmaceuticals or fine chemicals, inwhich the transgenic organism according to claim 12 is cultured and thedesired foodstuff, feedstuff, seed, pharmaceutical or fine chemical isproduced and/or isolated using said organism.
 18. The method of claim 1,wherein the transgenic expression cassette further comprises one or moregenetic control elements.
 19. The transgenic expression cassette ofclaim 7, wherein the expression cassette further comprises one or moregenetic control elements.
 20. A method for identifying and/or isolatinga sequence which directs expression in carbohydrate-storing sink tissue,but essentially not in source tissues comprising preparing fragments ofthe nucleic acid sequence of SEQ ID NO: 1; testing the fragmentsobtained for carbohydrate-storing sink tissue expression; andidentifying and/or isolating a fragment with carbohydrate-storing sinktissue expression activity.
 21. An expression cassette forcarbohydrate-storing sink tissue, but essentially not source tissues,expression in plants comprising at least one transcription regulatingnucleotide sequence, wherein the transcription regulating nucleotidesequence comprises a fragment obtained by the method of claim
 20. 22.The expression cassette of claim 21, further comprising at least onenucleic acid sequence which is operably linked to and heterologous inrelation to said transcription regulating nucleotide sequence, whereinthe transcription regulating nucleotide sequence directs expression ofthe further nucleic acid sequence in carbohydrate-storing sink tissue,but essentially not in source tissues.
 23. An expression cassette forcarbohydrate-storing sink tissue, but essentially not source tissues,expression in plants comprising i) at least one transcription regulatingnucleotide sequence, and ii) at least one further nucleic acid sequencewhich is operably linked to and heterologous in relation to saidtranscription regulating nucleotide sequence, wherein the transcriptionregulating nucleotide sequence comprises the nucleotide sequencedescribed by SEQ ID NOs: 1, 2, or 3, or a fragment thereof having thesame promoter activity as the nucleotide sequence of SEQ ID NO: 1, 2, or3; and wherein the transcription regulating nucleotide sequence directsexpression of the further nucleic acid sequence in carbohydrate-storingsink tissue, but essentially not in source tissues.
 24. A method fordirecting carbohydrate-storing sink tissue expression in a plantcomprising: I. introducing into a plant cell the expression cassette ofclaim 23, II. selecting a transgenic cell which comprise said expressioncassette, and III. regenerating a plant from the transgenic cell,wherein the transcription regulating nucleotide sequence directscarbohydrate-storing sink tissue, but essentially not in source tissues,expression of the operably linked nucleic acid sequence in the plant.